CN101611057B - Antibody molecules for human il-17 - Google Patents
Antibody molecules for human il-17 Download PDFInfo
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Abstract
Binding members, especially antibody molecules, for interleukin 17 (IL-17). The binding members are useful for the treatment of disorders associated with interleukin 17 such as rheumatoid arthritis.
Description
The present invention relates to the binding members, particularly antibody molecule of interleukin-17 (IL-17 is also referred to as IL-17A), and the application of their therapeutic, the IL-17 relative disease for example treated, such as rheumatoid arthritis.
IL-17A is the cell-derived cytokine of T-, and it is short scorching active that it has multiple-effect.External, IL-17A is adjusted to fibrocyte and the synovial cell produces inflammatory mediator, also can promote cartilage degradation and broken bone bone resorption with other pro-inflammatory cytokine synergy.
IL-17A is by 155 amino acid whose two homodimers that chain consists of.Each polypeptide chain contains 23 amino acid N-terminal peptide, its cutting is produced the mature polypeptide of 132 residues.IL-17A applies its effect (Toy etc., 2006) in conjunction with IL-17 acceptor A and C by activating the two.There are 5 kinds of homologues in IL-17A, is called IL-17B to IL-17F, and they all have distinct biological action.The Swissport accession number of IL-17 homologue is: IL-17B Q9UHF5; IL-17C Q96P0M4; IL-17D Q8TAD2; IL-17E Q9H293; IL-17F Q96PD4.The tissue expression of IL-17B and IL-17C is extensive, and the cell derived of this protein is not clear, although find the level of IL-17B transcript in neural system high (Moore etc., 2002).IL-17B and IL-17C can induce the TNF α (Li etc., 2000) of monocytic series THP-1, induce neutrophil to increase (Shi etc., 2000) when it is injected into mouse model.IL-17D detects IL-17D in Various Tissues, it is at dormancy CD4+T cell and CD19+B cells (Starnes etc., 2002).IL-17E (IL-25) causes the Th2 type and replys, for example airway hyperreactivity and eosinophilia, and its characteristic is different from other family member (Fort etc., 2001).There are two kinds of isotypes in IL-17F, it shows highest homology (55 with 40% homology) and has many similar functional performances to IL-17A, for example in lung, induce neutrophil to increase and induce pro-inflammatory cytokine, comprise IL-8 (Hymowitz etc., 2001; Hurst etc., 2002; Oda etc., 2005).The IL-17 family member may have effect in congenital and adoptive immunity, because only the antibody capable of target IL-17A guarantees to suppress to express the specific function of IL-17A signal conduction in the zone of IL-17A.
Combination between family member and the IL-17 receptor family is not understood fully.Known IL-17E can pass through the IL-17RB conducted signal, although also report IL-17B with than low-affinity in conjunction with (Lee etc., 2001).Except IL-17RA, report IL-17A in conjunction with IL-17RC (IL-17RL), similarly, IL-17F also with these receptors bind (Toy etc., 2006; Kuestner etc., 2005).Also have other acceptor, IL-17RD and IL-17RE are although their endogenic ligand not yet identifies.IL-17RC and IL-17RD exist multiple splice variant (Haudenschild etc., 2002; Haudenschild etc., 2006; Moseley etc., 2003).
The combination of IL-17A and its acceptor may be induced the acceptor oligomerization, thereby causes its activation.IL-17A activates IL-17RA and has activated many short scorching signal pathways, and for example extracellular signal is regulated protein kinase (ERK1/2), the terminal kinases (JNK) of c-jun N-and p38 map kinase approach.Activate these approach and trigger short scorching gene and the change of protein expression horizontal extent by not yet fully clear and definite mechanism.
IL-17A is mainly by CD4+ and CD8+T emiocytosis (Lubberts etc., 2001).In neutrophil, eosinophilic granulocyte and human blood mononuclear cell, also detect IL-17A, no matter its RNA or intracellular protein (Molet etc., 2001; Ferretti etc., 2003).Recently (discovery), IL-6 and TGF β relate to Naive T cells and are divided into the T cell (Betteli etc. that produce IL-17,2006), although other two kinds of cytokines relate to rheumatoid arthritis, and IL-15 and IL-23 regulate T lymphocyte release IL-17A.
IL-17A raises synovioblast and produces inflammatory cytokine and prostaglandin(PG), promotes synovioblast and articular chondrocytes to produce MMP, and it may have effect in broken bone bone resorption.With regard to the inflammatory environment in rheumatoid arthritis (RA) tissue, IL-17A may have and other pro-inflammatory cytokine, the particularly collaborative specific function of TNF-α and IL-1 β.
Several evidence prompting IL-17 play an important role in the pathogeny of rheumatoid arthritis (RA).Compare with 0/3 normal synovial membrane explant with 2/12 OA explant, functional IL-17A is by 16/18 RA synovial membrane explant Autocrine.The immunostaining of RA synovial membrane discloses the cell of generation IL-17 in the zone of being rich in the T cell (Chabaud etc., 1999).Compare the level of IL-17A rising (Cho etc., 2004) among the RA patients serum with normal control serum.
IL-17A shows the release that can stimulate a series of pro-inflammatory cytokines.IL-17A acts synergistically with other cytokine sometimes, strengthens synovioblast and produces IL-1, IL-6 and LIF (Katz etc., 2001; Chabaud etc., 1998).In addition, IL-17A shows that the COX-2 that can raise in the inflammatory cell expresses.
IL-17A induces COX-2 genetic expression (for example, Faour etc., 2003 in human chondrocytes, synovioblast and scavenger cell and the people's synovial membrane explant; Le Grand etc., 2001).Restructuring IL-17A raises the synovial cell COX-2 expression (Stamp etc., 2004) that synovial cell COX-2 expresses also enhance TNF α-stimulation.
Except these " classics " short scorching activity, IL-17A also causes other effect in the RA joint, for example promote cartilage degradation.IL-17 also shows participation MMP rise and affects cartilage degradation.IL-17A can increase the spontaneous generation MMP1 of people RA synovial cell.IL-17A raises MMP3, MMP13 and the ADAMTS4 gene (Sylvester etc., 2004) in the ox articular chondrocytes jointly.IL-17A is potent substrate degradation inductor and matrix synthetic inhibitor (Cai etc., 2001) in the joint cartilage explant.A kind of mechanism that IL-17A suppresses the articular chondrocytes synthetic substrate is to produce (Lubberts etc., 2000) by strengthening NO.Adenovirus IL-17A is injected into accelerates in the knee joint of II Collagen Type VI immune mouse and worsen synovial membrane inflammation, promote the destruction of joint (Lubberts etc., 2001) in this site.IL-17A also can participate in the broken bone bone resorption (Kotake etc., 1999) among the RA.
The importance of IL-17A biological characteristics is itself and other synergistic ability of cytokine.For example, IL-17A strengthens IL-1, IL-6 and the IL-8 synthetic (Katz etc., 2001) of TNF-α in the synovioblast-induce.In addition, in scleroblast system, MC-3T3, IL-17 and TNF-α show the synergy (Ruddy etc., 2004) of potent mediation IL-6 secretion.IL-17A enhancing IL-1 β-induce the synovial cell to produce IL-6 and LIF (Chabaud etc., 1998).
Evidence is further supported the effect of IL-17A in RA in the body of disease animal model.The overexpression of IL-17A the suis cell walls (SCW) of IL-1 deficient mice-induce induce in the sacroiliitis cartilage injury (Koenders etc., 2005b).In collagen-induced sacroiliitis (CIA) model, the overexpression of IL-17A promotes sacroiliitis (Chabaud and Miossec, 2001).In addition, IL-17A deficient mice or be subjected to suppress (Nakae etc., 2003 with CIA in the mouse of anti-IL-7mAb treatment; Lubberts etc., 2001; Lubberts etc., 2004).
Except its effect in RA, IL-17A is active relevant with many other pathology situations, comprising:
-osteoarthritis (for example, Honorati etc., 2002; Malemud etc., 2004);
-bone loss (Lubberts etc., 2003) and bone implant loosen (Van Bezooijen etc., 1999);
-air flue supersensitivity comprises allergic airway diseases, such as asthma (Molet etc., 2001; Wong etc., 2001; Linden, 2006) and ARDS;
-demyelination comprises multiple sclerosis (Lock etc., 2002; Touil etc., 2006);
-psoriatic (Teunissen etc., 1998) and psoriatic arthritis;
-corium supersensitivity comprises atopic dermatitis (Toda etc., 2003);
The rejection of-acute graft (Antonysamy etc., 1999; Yoshida etc., 2006; Tang etc., 2001);
-allograft refection (Hsieh etc., 2001);
-graft versus host disease (GVH disease);
-Sjogren's syndrome disease (Kurasawa etc., 2000);
-systemic lupus erythematosus (Wong etc., 2000);
-autoimmunization inflammatory bowel comprises ulcerative colitis (Nielsen etc., 2003; Fujino etc., 2003; Yen etc., 2006) and Crohn's disease;
-Urology Surgery inflammatory diseases comprises benign prostatic hyperplasia (Kramer and Marberger, 2006);
-cardiovascular disorder comprises atherosclerosis (Csiszar and Ungvari, 2004), mucocutaneous lymphnode syndrome (Sohn etc., 2003), ischemic heart disease (Csiszar, 2003) and apoplexy;
-vasculitis comprises behcet's disease (Hamzaoui etc., 2002);
-periodic fever comprises familial Mediterranean fever (Haznedaroglu etc., 2005);
-glucose metabolism comprises 1 type and diabetes B (Fisman, 2003);
-tuberculosis comprises chronic obstructive pulmonary disease (Shen etc., 2004a; Shen etc., 2004b), bronchitis, pulmonary emphysema, bronchiolitis obliterans (Vanaudenaerde etc., 2003) and pulmonary fibrosis;
-cancer comprises lymphoma (Maggio etc., 2002) and tumour (Numasaki etc., 2005);
-periodontopathy (peridontitis) (Vernal etc., 2005; Takahashi, 2005);
The disease that-virus infection causes comprises herpetic interstitial keratitis (herpetic stromal keratitis) (Maertzdorf etc., 2002);
The inflammatory diseases of-other IL-17 mediation for example comprises that anaphylaxis, reactive arthritis, inflammatory pain, spondyloarthropathy comprise ankylosing spondylitis, the inflammatory diseases of skin and cornea, inflammatory muscle disease;
The acute inflammatory response of-other IL-17 mediation for example comprises the infection of septicemia, septic shock and endotoxin shock, meningitis and wound (surgical operation);
The autoimmune disease of-other IL-17 mediation for example comprises, the autoimmunity blood disease, and alzheimer's disease, sarcoidosis, liver cirrhosis, gall-bladder and hepatic diseases comprise hepatitis, glomerulonephritis;
The Metabolic disorder of-other IL-17 mediation comprises dyslipidemia (dislipidemia).
In many arthritis models, blocking-up IL-17A inflammation-inhibiting, destruction of joint and progression of disease in the body.In Muridae CIA, utilize IL-17R Fc fusion rotein proof to suppress joint injury (Lubberts etc. at macroscopic scale, 2001), rat adjuvant induces that arthritic histologic analysis also proves this point (Bush etc., 2002) in sacroiliitis (AIA) model.In arthritic infectious model, utilize the commercialization neutralizing antibody proof for IL-17 (large mouse-anti-mouse) and IL-17R (large mouse-anti-mouse) can suppress swelling and sacroiliitis outbreak (Burchill etc., 2003).Rabbit is anti--and mouse monoclonal antibody has been used for arthritic murine model, and the severity that prove joint injury and cartilage destruction reduces and comprises the reduction of pro-inflammatory mediator level (Lubberts etc., 2004 of RANKL, IL-1 β and IL-6; Koenders etc., 2005a).
In and IL-17 proof in other animal model of a series of diseases, function is arranged.IL-17R Fc fusion rotein has prolonged survival time (Antonysamy etc., 1999) in the Mice Grafted with Allogeneic transplantation model.Induce in the mouse model of colitis at trinitro-benzene-sulfonic acid (TNBS), IL-17R IgG fusion rotein has also weakened colonic inflammation (Zhang etc., 2006).Commercialization monoclonal anti-IL-17 proves effect there are (Hellings etc., 2003) in mouse model by the segmental bronchus neutrophil that reduces behind the ovalbumin (Ova-challenge).In the rat model of surgery surgical adhesions (surgical adhesion), the rabbit polyclonal IL-17 of family neutralizing antibody also reduces adhesion in the dose-dependently mode (adhesiogenesis) (Chung etc., 2002) occurs.
Antibody for IL-17A has been seen description.For example, (the R ﹠amp of RD system house; D Systems) having produced Muridae resists-IL-17 monoclonal antibody MAB317.Develop this IgG2B antibody from the Mouse Hybridoma Cells that recombinant human IL-17 (intestinal bacteria derive) immune mouse of purifying causes.In addition, now developed have in and another Muridae of characteristic anti--IL-17 antibody 53.159.16 (Biological resources company (Biosource Inc.)).Develop this IgG1K antibody from the Mouse Hybridoma Cells that the recombinant human IL-17 immune mouse (Balb/c) of purifying causes.Natural and the restructuring IL-17 of this antibody recognition.
WO2006/054059 (UCB Cell Technology Inc. (UCB CellTech)) has described the neutralizing antibody molecule of IL-17A.Described at first from hybridoma separate anti--IL-17 antibody is all is the PEGization antibody fragment that form is derived.The document claims that adopting BIAcore can measure this antibody fragment is 133-365pM to the avidity of IL-17.
WO2006/013107 (Novartis limited-liability company (Novartis Pharma GmbH)) has described the binding members of IL-17A, particularly is called AIN457, and the people who separates from hybridoma resists-IL-17 IgG1 antibody.WO2006/013107 has reported the BIAcore avidity detected value (Kd) of the IL-17 interaction of restructuring AIN457 and people's (0.227nM), marmoset (1.2nM), macaque (9nM) and stump-tailed macaque (6nM), shows that the avidity to people and stump-tailed macaque IL-17 differs respectively about 25 times.
This paper describes the binding members of IL-17A, be also referred to as the IL-17A-binding members.Binding members of the present invention can be the antibody molecule of IL-17A, particularly human antibody molecules.
These binding members can be used for treating the IL-17A relative disease, and the IL-17-relative disease that claims of its place of one or more this paper for example is such as rheumatoid arthritis.
This paper describes the binding members in the zone, 71-87 position that is combined in ripe human il-17 A.The sequence of 71-87 position is: Cys-Arg-His-Leu-Gly-Cys-Ile-Asn-Ala-Asp-Gly-Asn-Val-Asp-Tyr-His-Met (SEQ ID NO:199).
Binding members of the present invention can be in conjunction with at least one residue among the Cys-Arg-His-Leu-Gly-Cys-Ile-Asn-Ala-Asp-Gly-Asn-Val-Asp-Tyr-His-Met (SEQ ID NO:199).For example, it can be in conjunction with the residue more than 1,2,3,4,5 or 5 among the SEQ ID NO:199.
Binding members of the present invention can be in conjunction with residue Leu74, Tyr85, His86 and/or the Met 87 of the human il-17 A (SEQ ID NO:198) of maturation.For example, it can be in conjunction with Leu74, Tyr85 and/or His86.Binding members can be in conjunction with all these residues.Can pass through, the specificity interaction (for example at binding members: in the structure of IL-17A mixture) that for example detects or observe between binding members and the IL-17A residue is measured in conjunction with situation, can adopt such as the X-radiocrystallography and measure.The structure that adopts the X-radiocrystallography to measure the antibody 7 of being combined with human il-17 A shows that antibody 7 is in conjunction with residue 74 (Leu), 85 (Tyr), 86 (His) and 87 (Met) of mature sequence (SEQ ID NO:198).
Except in conjunction with the one or more residues in the SEQ ID NO:199, binding members can be chosen wantonly in conjunction with the side joint residue in the IL-17A molecule or the residue that structurally adjoins.For example, binding members can be in conjunction with Asn88.
Binding members of the present invention can be in conjunction with the one or more following residue among the human il-17 A (SEQ ID NO:198) of maturation, and is for example 5 or more, for example 10 or more, for example whole:
Ser40
Ser41
Asp42
Tyr43
Arg46
Leu74
Tyr85
His86
Met87
Asn88
Pro126
Ile127。
Binding members of the present invention can be in conjunction with residue Ser40, Ser41, Asp42, Tyr43 and/or the Arg46 of the human il-17 A of maturation.
Binding members of the present invention can be in conjunction with residue Tyr85, His86, Met87 and/or the Asn88 of the human il-17 A of maturation.
Binding members can be in conjunction with Pro126 and/or the Ile127 of the human il-17 A of maturation.
State such as its place of this paper, IL-17A is formed with the dimer of two polypeptide chains.Binding members of the present invention or VH structural domain can be in conjunction with the IL-17A polypeptide of arbitrary maturation in the dimer.
For example, binding members can be in conjunction with Tyr85, His86, Met87 and/or the Asn88 of an IL-17A polypeptide in the dimer, and/or can be in conjunction with Pro126 and/or the Ile127 of same polypeptide, and/or can be in conjunction with Ser40, Ser41, Asp42, Tyr43 and/or the Arg46 of another IL-17A polypeptide in the dimer.
Can adopt any suitable method, for example hydrogen-deuterium exchange, site-directed mutagenesis, mass spectroscopy, NMR and X-radiocrystallography are measured the residue sequence of binding members institute combination.
Those skilled in the art know and adopt the X-radiocrystallography to measure the accurate three-dimensional structure of protein with atom definition, also can adopt the X-radiocrystallography to estimate in detail in the protein and the interactional part of antibody (Padavattan etc., 2007; Karpusas etc., 2001).In case obtain to form with target antigen the crystal of the binding members of mixture, their diffraction patterns to obtain to depend on that definite atom distributes of available x-ray irradiation.Can utilize crystallograph (crystallographer) to analyze diffraction pattern to measure the three-dimensional location coordinates of each atom in the structure.Thereby can detect in detail the interaction sites between binding members and the IL-17A.
Therefore, can measure the IL-17A residue of being combined with binding members by the interaction between binding members and the IL-17A residue in the X-ray crystal structure of observing the binding members of being combined with IL-17A.For example, in conjunction with comprising that hydrogen bond and/or nonpolar (hydrophobicity) interact.Can adopt 3.2
Block the distance measure hydrogen bond, utilize 4.0
The distance of blocking measure apolar interaction.Measure and the example identified is shown in the embodiment 7.4 of this paper in conjunction with the X-ray crystal structure of residue.
The exchange of peptide amide hydrogen is the well-known process (Englander etc., 1994) for Study on Protein.Recently, further developed the deuterium-labeled protein that proton exchanges, the exchange speed (Pantazatos etc., 2004) to detect whole protein with the method and mass spectroscopy coupling done of utilizing.Can utilize accessibility with solvent obviously to change hydrogen/deuterium and exchange (H/D exchanges) speed, thus when certain subparticipation of protein in conjunction with another minute period of the day from 11 p.m. to 1 a.m, exchange speed is reduction obviously.The method be used for have been drawn protein and has been related to collection of illustrative plates (Lu etc., 2005) with the interactional zone of antibody, and is used for research IL-17A and relates to zone with antibodies of the present invention, as described in this paper embodiment 7.1.The coupling mass spectroscopy exchanges to identify the zone that contacts binding members among the IL-17A with H/DA.For example, can prove when IL-17A when contact member of the present invention is combined, the H/D of residue exchanges and obviously slows down in the SEQ IDNO:199.
Those skilled in the art are known as structure are interrelated and single amino acids and a plurality of zone of mutagenesis protein with active, and the method is for determining the zone (Lu etc., 2005) of protein with antibodies.Adopt these technology to see embodiment 7.2 in conjunction with the example of binding members of the present invention.
Can adopt and be combined with the mutant human IL-17A with aminoacid sequence shown in the SEQ ID NO:201 and/or assess whether combination in the zone of 71-87 position among the human il-17 A in maturation of binding members with its neutralization.Compare with wild-type, mutant IL-17A do not exist in conjunction with or neutralizing effect, perhaps in conjunction with or neutralizing effect significantly reduce, show that binding members is combined with at least one residue of SEQ ID NO:199.
Binding members of the present invention can choose wantonly be not combined with the mutant human IL-17A with aminoacid sequence shown in the SEQ ID NO:201 and/or with its neutralization.For example, not mutation inhibiting type human il-17 A (SEQ ID NO:201) and its receptors bind of binding members of the present invention.Can be by the neutralizing effect of various test determination IL-17A, it locates to have described the example of described test this paper.
Binding members of the present invention can not suppress the IL-6 release that mutant human IL-17A in the HT1080 cell (SEQ ID NO:201) induces.In the HT1080 cell IL-6 release test of the mutant IL-17A that utilizes concentration for 2nM (SEQ ID NO:201), binding members of the present invention can not show that obvious neutralization renders a service, and for example binding members member's concentration is in the highest 50nM.
In the IL-6 of HT1080 cell release test, the IC50 Billy who utilizes binding members of the present invention in the test of 1nM mutant human IL-17A (SEQ IDNO:201) is with in the test of the human il-17 A (SEQ ID NO:198) of 1nM or 2nM maturation high 10 times, high 20 times, high 50 times or high 100 times.Instance data is shown in embodiment 7.3 (table 20b), shows that the effectiveness of 7 couples of mutant IL-17A of antibody is far below (IC50 is higher) the human il-17 A to maturation.Therefore should be noted that the IC50 value of in the test with mutant IL-17A, failing to measure antibody 7, show that any IC50 value is higher than that this test can detect.
State such as its place of this paper, can adopt surperficial plasmon resonance, for example BIAcore measures the combination of binding members and IL-7.For example, binding members of the present invention can show and the not obvious combination of mutant human IL-17A with aminoacid sequence shown in the SEQ ID NO:201, for example in surperficial plasmon resonance test less than about 10RU.
Can be according to divalence analyte model (simultaneously ka kd) fit surface plasmon resonance data and the affinity constant Kd that calculates from the ratio kd1/ka1 of velocity constant.Binding members of the present invention in conjunction with the avidity of ripe human il-17 A (SEQ ID NO:198) than high 5 times in conjunction with the binding affinity of mutant human IL-17A (SEQ ID NO:201), for example high 10 times or high 50 times.
Also can adopt the IL-17A residue such as technical evaluation binding members institute combinations such as X-radiocrystallographies, can adopt these technology to confirm and/or optimize the result of other technology, for example identify the residue that contacts with binding members.Those skilled in the art know the X-radiocrystallography that adopts protein and measure secondary, three grades and quaternary structure, this technology are used for drawing collection of illustrative plates (Padavattan etc., 2007 of protein and the interactional part of antibody; Karpusas etc., 2001).The isolated polypeptide or the peptide that comprise aminoacid sequence shown in the SEQ ID NO:199, or the peptide of the separation that is made of aminoacid sequence shown in the SEQ ID NO:199 can be used for producing, separate and/or testing other IL-17A binding members of the present invention.
Therefore, other side of the present invention relates to the isolated fragment of ripe human il-17 A sequence (SEQ ID NO:198), and these fragments comprise aminoacid sequence shown in the SEQ ID NO:199 or are made of it.For example, these fragments can reach at most 20,25,30,50 or 100 amino acid.Can comprise one or more fragments in longer peptide or the peptide sequence, described peptide or peptide sequence are not the IL-17A aminoacid sequences, for example are not precursor or ripe IL-17A, for example are not SEQ ID NO:198.Comprise the isolated fragment of IL-17A aminoacid sequence or peptide or the polypeptide of a plurality of fragments and can comprise other amino-acid residue, wherein said other residue does not adjoin with the IL-17A aminoacid sequence.For example, after the SEQ ID NO:199 or can be the one or more residues that do not adjoin with SEQ ID NO:198 before.The binding members of this peptide species or peptide, for example antibody molecule belongs to the present invention.
Endogenous IL-17A in the body is through glycosylation, therefore, and the therapeutic target antigen that glycosylated human il-17 A is human therapy.Although bacterial derivation (for example, at expression in escherichia coli) and can be used for test described herein without glycosylated recombinant human IL-17A, but we prove that also binding members of the present invention can be in conjunction with glycosylated human il-17 A, for example the IL-17A that produces of human T-cell or human embryo kidney (HEK) (HEK) EBNA cell.This has embodied the obvious advantage of binding members of the present invention, because glycosylated human il-17 A is the target antigen of human in the body.Binding members of the present invention can be roughly the same to avidity and/or the effectiveness of glycosylation and non-glycosylated IL-17A.
Be described in more detail among the embodiment binding members of the present invention can be efficient in and IL-17A.The neutralization expression suppresses the biologic activity of IL-17A.Binding members of the present invention can in and one or more biologic activity of IL-17A.The biologic activity that suppresses is the combination of IL-17A and its one or more its binding partners or acceptor normally, for example with the combination of IL-17RA.
Can be by biological molecule in the biological test, for example MMP13, PGE2 or cytokine, detect the neutralizing effect of IL-17 and a receptors bind such as the cell release conditions of IL-6 or IL-8, because IL-17A and its receptors bind induce the cell of these molecules to discharge, thereby can adopt suitable test, for example in the cell or tissue of HT1080 cell (ECACC number 85111505), chondrocyte or other adequate types, measure.
Can partially or completely suppress biologic activity.In embodiment, compare with the activity under not having the binding members existence, the binding members that provides can suppress at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50% with the IL-17 biologic activity.Degree with IL-17A in the binding members is called its neutralization effectiveness.Can adopt those skilled in the art one or more test determinations or detection known or described herein or that address to render a service.For example, can be in following test testing effect:
-HT1080 IL-6 release test
-HT1080 the test cell line that utilizes collaborative IL-6 that IL-17 and TNF α are reacted to discharge
-chondrocyte IL-6/IL-8/MMP13/PGE2-release test
IL-6 test in the-cartilage explant
IL-6 release test in the-synovioblast (for example, RA or OA patient's) for example, utilizes the collaborative IL-6 reaction for IL-17 and TNF α.
Test method is described in embodiment.
Can will (for example utilize the first species, the neutralization of the binding members that calculates in the test of the IL-17 people) is renderd a service with this binding members and (is for example being utilized the second species, stump-tailed macaque) neutralization in the identical test of IL-17 is renderd a service and is made comparisons, thereby assesses the cross reaction degree of the IL-17 of this binding members and two kinds of species.
Binding members of the present invention is in conjunction with human il-17 A and stump-tailed macaque IL-17A, in it and the effectiveness difference of people and stump-tailed macaque IL-17A less than 30 times, for example less than 25,20,15,10,5 or 2 times, for example in HT1080 IL-6 release test, measure.
For example, in HT1080 IL-6 release test described herein, the data of this paper show in 1-4 number and the 7-16 antibody and the effectiveness difference of people and stump-tailed macaque IL-17A respectively less than 20 times.Data are shown in embodiment 2.Therefore, in some embodiments, binding members of the present invention is renderd a service in 20-times the neutralization of people and stump-tailed macaque IL-17A.In some embodiments, roughly the same or equivalent to the neutralization effectiveness of people and stump-tailed macaque IL-17A, namely in 10-times.
Unless statement is arranged in addition, renders a service the IC that is typically expressed as in nM
50Value.In function test, IC
50Biologically to be reduced by 50% binding members concentration from its maximum.Can by draw the functional arrangement of the highest biologically % and log (binding members concentration), utilize such as the Prism of figure pad company (GraphPad) or the software programs such as Origin in origin laboratory (Origin Labs) and calculate IC
50Thereby, can be according to the data fitting sigmoid function to produce IC
50Value.
At human il-17 A as herein described
In the test, the neutralization of binding members of the present invention is renderd a service or IC
50Be up to 15nM.Can adopt the IC of the binding members of this test determination scFV form
50For example, IC
50The highest can be 10.0,5.0,4.0,3.0,2.0 or 1.0nM.Exemplary IC
50Data are seen embodiment 2 (referring to table 5).
Receptor-ligand is 0.75nM in conjunction with the final concentration of human il-17 A used in the test, and method detailed is seen embodiment.
In human il-17 HT1080 IL-6 release test, the neutralization of binding members of the present invention is renderd a service or IC
50Be up to 40nM.For example, IC
50The highest can be 35,25,20,15,10 or 5nM.In human il-17 HT1080 IL-6 release test, the neutralization of binding members of the present invention is renderd a service or IC
50Be up to 3.0,2.0 or 1.0nM.Exemplary IC
50Data are seen embodiment 2 (referring to table 6A).This test detects the IL-6 that 1nM human il-17 A is reacted and discharges, and method detailed is seen embodiment.
In human il-17 HT1080 IL-6 release test, the pA of binding members of the present invention
2Value is about 10 or less than 10.PA
2The method of analyzing is described in the embodiment chapters and sections.Example data is seen embodiment 3 (referring to table 8a).
In detecting the collaborative HT1080 test cell line that discharges of IL-6 that 125pM human il-17 A and 25pM TNF α are reacted, the neutralization of binding members of the present invention is renderd a service or IC
50Be no more than 1nM.In this test, the neutralization of binding members of the present invention is renderd a service or IC
50Be no more than 1,0.5 or 0.3nM.Exemplary IC
50Data are seen embodiment 2 (referring to table 6b).
In the HT1080 cell that human T-cell's glycosylation IL-17A stimulates, binding members of the present invention suppresses the IC that IL-6 discharges
50Be no more than 1nM, for example be no more than 0.9,0.8,0.7,0.6,0.5,0.4,0.3,0.2,0.1nM.Exemplary IC
50Data are seen embodiment 2 (referring to table 6c).These data show antibody of the present invention can in conjunction with and in and natural human IL-17A.
Preferred combination senior middle school and effectiveness and good species cross reactivity.Therefore, for example discharge in the HT1080 test at IL-6, the IC50 of binding members and human il-17 A is no more than 1nM, wherein said IC50 in human il-17 A HT1080 test and the IC50 of stump-tailed macaque IL-17A HT1080 in testing differ less than 10 times.The neutralization that can be higher than stump-tailed macaque IL-17A is renderd a service in the neutralization of human il-17 A to be renderd a service.
In stump-tailed macaque IL-17 HT1080 IL-6 release test, the neutralization of binding members of the present invention (for example IgG1 form) is renderd a service or IC
50Be up to 150nM.For example, IC
50The highest can be 150,100,50,40,30,25,20,15,10 or 5nM.Exemplary IC
50Data are seen embodiment 2 (referring to table 6A).The final concentration of used stump-tailed macaque IL-17A is 1nM in the IL-6 release test of HT1080 cell, and method detailed is seen embodiment.
Can adopt test determination as herein described to suppress the effectiveness that IL-6, IL-8, MMP13 and/or PGE2 discharge among the former generation chondrocyte of people.In these tests, the example data of binding members of the present invention is seen embodiment 4.In the chondrocyte IL-6 of the IL-17 that utilizes 0.2nM concentration release test, the IC of binding members of the present invention
50Be up to 3.0nM, for example be up to 2.0,1.0,0.9,0.8,0.7,0.6,0.5,0.4,0.3 or 0.2nM.In the chondrocyte IL-6 of the IL-17 that utilizes 2nM concentration release test, the IC of binding members of the present invention
50Be up to 20nM, for example be up to 10,5.0,4.0,3.0 or 2.0nM.
In the chondrocyte IL-8 of the IL-17 that utilizes 0.2nM concentration release test, the IC of binding members of the present invention
50Be up to 3.0nM, for example be up to 2.5,2.3,1.0,0.5,0.4,0.3,0.2 or 0.1nM.In the chondrocyte IL-8 of the IL-17 that utilizes 2nM concentration release test, the IC of binding members of the present invention
50Be up to 8nM, for example be up to 5,4,3,2,1.8,1.6,1.5,1.0 or 0.8nM.
In the chondrocyte MMP13 of the IL-17 that utilizes 0.2nM concentration release test, the IC of binding members of the present invention
50Be up to 5nM, for example be up to 4,3,2,1,0.5nM.In the chondrocyte MMP13 of the IL-17 that utilizes 2nM concentration release test, the IC of binding members of the present invention
50Be up to 40nM, for example be up to 30,20,10,5nM.
In the chondrocyte PGE2 of the IL-17 that utilizes 2nM concentration release test, the IC of binding members of the present invention
50Be up to 6nM, for example be up to 5,4,3,2,1,0.5nM.
In the after death cartilage explant IL-6 of the IL-17A that utilizes 10ng/ml concentration and 1ng/ml TNF α concerted reaction release test, binding members of the present invention can show that inhibition is active, and for example at least 80% or 90% suppresses, or 100% suppresses.In the after death cartilage explant IL-6 of the IL-17A that utilizes 10ng/ml concentration and 1ng/ml TNF α concerted reaction release test, the IC of binding members of the present invention
50Value is no more than 5nM, for example is no more than 4,3,2,1,0.5nM.Example data is seen embodiment 6.1 (table 15a and 15b).
In the IL-17/TNF of OA synovioblast as herein described α Synergism Testing (for example, in the IL-8 release test that utilizes 10ng/ml or 1ng/ml IL-17 and TNF α concerted reaction, wherein maximum reaction has IL-17 and TNF α component), binding members of the present invention can show that inhibition is active, and for example at least 50,60 or 65% suppresses.Example data is seen embodiment 6.2 (table 16a and 16b).
In the synovioblast IL-8 of the IL-17 that utilizes 2nM concentration release test, the IC of binding members of the present invention
50Value is no more than 5nM, for example is no more than 4,3 or 2nM.Example data is seen embodiment 6.3 (table 17).
Add in the collaborative RA synovioblast IL-8 release test of TNF α the IC of binding members of the present invention at the IL-17A of the TNF of the IL-17 that utilizes 1nM concentration and 0.1ng/ml α
50Value is no more than 5nM, for example is no more than 4,3 or 2nM.Example data is seen embodiment 6.3 (table 17).
In pre-mixing mouse air bag as herein described (airpouch) test, IL-6 that binding members of the present invention can be induced IL-17A (mixing with binding members in advance) discharges and suppresses at least 25%, and for example at least 30,35,40,45,50,55 or 60%.Inhibition can be about 100%.For example, the ID of binding members in this test
50Be up to 10, for example be up to 5,4,3,2,1 or 0.5 μ g.In mouse pre-mixing air bag as herein described test, the white corpuscle flux that binding members of the present invention also can be induced IL-17A suppresses at least 80%, and for example at least 85,90,95,98 or 99%.Inhibition can be about 100%.For example, the ID of binding members in this test
50Be up to 5,4,3,2 or 1 μ g.The example data of binding members of the present invention (comprising 7 groups of the antibody 2 of CDR and antibody) is seen embodiment 5 (referring to table 13a and table 14a).
In general administration mouse air bag as herein described test, the IL-6 that binding members of the present invention can be induced IL-17A (general gives binding members, then gives air bag with IL-17A) discharges and suppresses at least 70%, and for example at least 75,80 or 85%.For example, the ID of binding members in this test
50Be up to 1 μ g, for example be up to 0.5,0.4,0.3,0.2,0.1 or 0.05 μ g.In mouse general administration air bag as herein described test, the white corpuscle flux that binding members of the present invention also can be induced IL-17A suppresses at least 70%, and for example at least 75,80,85,90 or 95%.Inhibition can be about 100%.For example, the ID of binding members in this test
50Be up to 2, for example be up to 1.5,1,0.9,0.8,0.7 or 0.6 μ g.The example data of binding members of the present invention (comprise CDR antibody 7 groups) is seen embodiment 5 (referring to table 13b and table 14b).
When the digital proof part that embodiment 5 described air bag tests obtain or general administration, binding members suppresses the ability of IL-17A induced reaction.These data show, give the IL-17A relative disease that binding members of the present invention can effectively be treated the inflammation part, therefore can be used for the treatment of such as diseases such as rheumatoid arthritiss by the IL-17A induced reaction that suppresses in the synovial joint.Should be noted that general gives the effect that binding members of the present invention shows energy establishment IL-17A, establishment need not topical.This is for the clinical application of binding members, and it is especially favourable for example to be used for human body, can adopt the general administration in this case, and the position of administration is normal different from the one or more positions that show disease, and one or more positions of inflammation for example are such as synovial joint.
In addition, can measure the IL-17A binding members (is expressed as equilibrium dissociation constant, Kd), for example adopts surperficial plasmon resonance, such as BIAcore, perhaps can pass through pA the binding kinetics of IL-17A and avidity
2Analytical estimating Kd.
For example, from the pA of human il-17 HT1080 IL-6 release test
2Analysis meter calculate binding members of the present invention to the Kd of IL-17A less than 1000pM, for example less than 600,500,400,300 or 200pM.
State such as its place of this paper, the resonance of surperficial plasmon comprises makes analyte flow through the part that links to each other with upholder in liquid phase, and between determination and analysis thing and the part in conjunction with situation.For example, can implement plasmon resonance so that IL-17A flows through the binding members that links to each other with upholder in liquid phase.Can be according to divalence analyte data model or unitary analysis thing data model fit surface plasmon resonance data.As described in the embodiment chapters and sections of this paper, find that divalence analyte data model is particularly suited for measuring the avidity of binding members and IL-17.Can adopt divalence analyte data model by the ratio kd1/ka1 of surperficial plasmon resonance finding speed constant, calculate affinity constant Kd by this velocity constant.
The exemplary IL-17 that adopts surperficial plasmon resonance to calculate sees embodiment 3 (referring to table 8b) in conjunction with the KD estimated value.7 pairs of binding characteristics at the recombinant human IL-17A of intestinal bacteria or the generation of HEK EBNA cells of these digital proof antibody are good.This antibody capable of proof of being combined with the IL-17A of HEK EBNA cell is combined with Natively glycosylated human il-17 A.Antibody 7 shows that in conjunction with Natively glycosylated form antibody 1-16 all can be in conjunction with Natively glycosylated human il-17 A, suppose that these antibody are all derived from a kind of parental generation antibody (antibody 1), therefore, believe they all can with the identical of IL-17A or highly similar epi-position combination.
The human il-17 A that binding members of the present invention is combined in people's cells (for example, the IL-17A that HEK EBNA-derives) with (for example be combined in the human il-17 A that expresses in the bacterial cell, the difference of the avidity IL-17A that intestinal bacteria derive) is less than 5 times, for example less than 2.5 times or less than 2 times.
Can adopt divalence analyte data model by the ratio kd1/ka1 of surperficial plasmon resonance finding speed constant, the binding members of the present invention that is calculated by this velocity constant is about the Kd of human il-17 A or less than 2.5nM.For example, adopt divalence analyte data model by the ratio kd1/ka1 of surperficial plasmon resonance finding speed constant, the binding members of the present invention that is calculated by this velocity constant to the Kd of human il-17 A less than 2.5nM, 2nM, 1.5nM, 1.0nM or 0.5nM.
Adopt unitary analysis thing data model to measure by surperficial plasmon resonance that binding members of the present invention is about the avidity of human il-17 A or less than 0.3nM.For example, adopt unitary analysis thing data model by surperficial plasmon resonate calculating book invention binding members to the Kd of human il-17 A less than 300pM, for example less than 200pM.
Adopt divalence analyte data model by the ratio kd1/ka1 of surperficial plasmon resonance finding speed constant, the binding members of the present invention that is calculated by this velocity constant is about the Kd of stump-tailed macaque IL-17A or less than 1.5nM.
Adopt unit price or divalence model-fitting data, adopt surperficial plasmon resonance be measured to binding members of the present invention to the avidity difference of people and stump-tailed macaque IL-17A less than 10 times.Therefore, adopt the resonance of surperficial plasmon be measured to binding members of the present invention in conjunction with the Kd of stump-tailed macaque IL-17A with in conjunction with the difference of human il-17 A less than 10 times, for example difference is less than 5 times or less than 3 times.State such as its place of this paper, avidity can be the Kd from the ratio kd1/ka1 calculating of velocity constant, and described velocity constant adopts divalence analyte data model to measure by surperficial plasmon resonance.Shown in embodiment 3 and table 8b, it is good in conjunction with the cross reactivity of people and stump-tailed macaque IL-17A to be measured to antibody 7.
Can utilize the cross reactivity of all antibody 1-16 of cross reactivity data representation of the antibody 7 of acquisition, suppose all these antibody derived from a kind of parental generation antibody (antibody 1), therefore believe they all can with the identical of IL-17A or highly similar epi-position combination.
The data of this paper report show binding members of the present invention not with IL-17 homologue, i.e. IL-17B, IL-17C, IL-17D, IL-17E or IL-17F cross reaction.Observe in some cases with IL-17F a little less than combination.For example, can measure cross reactivity by the degree that detection IL-17 homologue inhibition IL-17A is combined with binding members of the present invention, for example adopt as herein described
The epi-position competition experiments.As described herein, this test in the not obvious inhibition of IL-17 homologue B-E IL-17A be combined with binding members of the present invention, thereby prove these binding members not with IL-17 homologue B-E cross reaction.
In utilizing the epi-position competition experiments of IL-17F, binding members demonstration of the present invention is combined with IL-17A and suppressed by part (that is, less than 100%), and for example wherein the concentration of IL-17F is 1 μ M or higher.For example, IL-17F can suppress IL-17A in conjunction with being no more than 50%, for example is no more than about 20%.
None can suppress IL-17B, C, D, E or F binding members fully and be combined with human il-17 A.In the epi-position competition experiments, the IL-17B of 1 μ M, IL-17C, IL-17D, IL-17E or IL-17F (individually) all can not suppress binding members of the present invention fully is combined with human il-17 A.Therefore, in utilizing the epi-position competition experiments of mark IL-17A, the combination that the IL-17B of 1uM, IL-17C, IL-17D, IL-17E or IL-17F all can not suppress binding members and human il-17 A surpasses 50%, for example be no more than 20%, the concentration of described mark IL-17A equals binding members and the interactional dissociation constant Kd of human il-17 A, wherein adopt divalence analyte data model by surperficial plasmon resonance, the ratio kd1/ka1 of BIAcore finding speed constant for example, and calculate described Kd by this velocity constant.For example, in this epi-position competition experiments, the IL-17B of 1 μ M, IL-17C, IL-17D or IL-17E can not suppress binding members is combined with human il-17 A, and 1 μ MIL-17F may not suppress, and perhaps can suppress but less than 50%.
The example data of epi-position competition experiments is shown in embodiment 2.7 (referring to table 7).In utilizing the epi-position competition experiments of IL-17B, C, D, E or F, can not measure the IC50 value, show that too high so that this test of IC50 value can not detect.
Binding members of the present invention shows that the cross reactivity to the IL-17 homologue has the therapeutic and/or the diagnostic application that are limited to them that advantage is provided, and particularly needs interior application of body of specific inhibition of IL-6-17A.Reduced the side effect due to the bad cross reactivity.In addition, can utilize the lower binding members of concentration, the binding members of multiple doses comes in conjunction with target IL-17A because the cross reactivity finite presentation can give more.This especially has superiority for interior therapeutic application administration.
Binding members of the present invention can comprise antibody molecule, for example human antibody molecules.Binding members comprises antibody VH and/or VL structural domain usually.The VH structural domain of binding members also provides as part of the present invention.Complementary determining region (" CDR ") and framework region (" FR ") in each VH and the VL structural domain.The VH structural domain comprises one group of HCDR, and the VL structural domain comprises one group of LCDR.Antibody molecule can comprise the antibody VH structural domain that contains VH CDR1, CDR2 and CDR3 and framework.Perhaps it also can comprise the antibody VL structural domain that contains VL CDR1, CDR2 and CDR3 and framework.Between comprising shown in following structure, VH or VL structural domain framework be inserted with 4 framework regions of CDR, FR1, FR2, FR3 and FR4:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
Antibody VH of the present invention and VL structural domain, the example of FR and CDR are seen the sequence table of formation this paper part of enclosing.Other exemplary CDR sees below and table 21.All VH and VL sequence, the CDR sequence that this paper discloses, organize CDR more and organize HCDR and many group LCDR are representing aspects of the present invention and embodiment more." one group of CDR " as herein described comprises CDR1, CDR2 and CDR3.Therefore, one group of HCDR refers to HCDR1, HCDR2 and HCDR3, and one group of LCDR refers to LCDR1, LCDR2 and LCDR3.Unless statement is arranged in addition, " one group of CDR " comprises HCDR and LCDR.Binding members of the present invention is monoclonal antibody normally.
Another aspect of the present invention is the antibody molecule that comprises the VH structural domain and/or comprise the VL structural domain, any VH structural domain has at least 80,85,90,95,98 or 99% aminoacid sequence homogeny among described VH structural domain and the 1-16 of antibody shown in the sequence table that encloses, and any VL structural domain has at least 80,85,90,95,98 or 99% aminoacid sequence homogeny among described VL structural domain and the 1-16 of antibody shown in the sequence table that encloses.The algorithm that can be used for calculating the homogeny % of two seed amino acid sequences comprises, such as BLAST (Altschul etc., 1990), FASTA (Pearson and Lipman, 1988) or Smith-Waterman algorithm (Smith and Waterman, 1981), for example adopt default parameters.
Binding members of the present invention can comprise the intramolecular antigen binding site of non-antibody, is usually provided by the one or more CDR in the non-antibody protein scaffolds, and for example HCDR3 and/or LCDR3, or one group of CDR hereinafter will further discuss.
Such as the embodiment part in greater detail, we separate the parental generation antibody molecule (No. 1 antibody) that contains one group of CDR sequence shown in table 21.By optimizing, we produce one group of antibody cloning of 2-16 number, and its CDR3 sequence has replacement derived from parental generation CDR3 sequence and in position shown in the table 21.Therefore, for example as can be seen from Table 21, antibody 2 has parental generation HCDR1, HCDR2, HCDR3, LCDR1 and LCDR2 sequence, and has parental generation LCDR3 sequence, and wherein Kabat residue 93 and 94 is substituted by P and H respectively.Parental generation antibody molecule as herein described and antibody molecule 2-16 refer to respectively to contain the parental generation antibody molecule CDR antibody molecule and contain the antibody molecule of the CDR of antibody 2-16.
This paper describes the binding members that comprises the group of parental generation shown in the table 21 CDR, wherein HCDR1 is that SEQID NO:3 (Kabat residue 31-35), HCDR2 are that SEQ ID NO:4 (Kabat residue 50-65), HCDR3 are that SEQ ID NO:5 (Kabat residue 95-102), LCDR1 are that SEQ ID NO:8 (Kabat residue 24-34), LCDR2 are that SEQ ID NO:9 (Kabat residue 50-56) and LCDR3 are SEQ IDNO:10 (Kabat residue 89-97).
Binding members of the present invention can comprise one or more CDR described herein, and CDR3 for example also optionally comprises CDR1 and CDR2, thereby forms one group of CDR.CDR or CDR group can be parental generation CDR or parental generation CDR group, perhaps can be that any CDR or CDR organizes among the antibody 2-16, perhaps can be its variant, and be as described herein.
For example, binding members of the present invention or VL structural domain can comprise parental generation LCDR3, and wherein Kabat residue 93 and 94 is substituted by P and H respectively.
Binding members or VH structural domain can comprise and contain one or more following parental generation HCDR3 that replace:
The Kabat residue 98 that F or H substitute;
The Kabat residue 100A that G or T substitute;
The Kabat residue 101 that R substitutes;
The Kabat residue 102 that G or N substitute.
Binding members obtains the VL structural domain can comprise the parental generation LCDR3 with one or more following replacements:
The Kabat residue 90 that T substitutes;
The Kabat residue 92 that N or S substitute;
The Kabat residue 93 that H or P substitute;
The Kabat residue 94 that H, K, R, T or Y substitute;
The Kabat residue 95 that D, N or V substitute;
The Kabat residue 96 that I or Q substitute.
The D that replaces Kabat position 93 among the LCDR3 with P is relevant to the effectiveness enhancing of people and/or stump-tailed macaque IL-17A with binding members.
Binding members of the present invention can comprise among the antibody 1-16 any HCDR1, HCDR2 and/or HCDR3 and/or antibody 1-16 in any LCDR1, LCDR2 and/or LCDR3, the one group of CDR of any among the example antibody 1-16 shown in table 21.Binding members can comprise one group of VH CDR of one of these antibody.It can choose the one group of VL CDR that comprises one of these antibody wantonly, and described VL CDR can be from the antibody identical or different with VH CDR.Comprise among the antibody 1-16 any one group of HCDR and/or antibody 1-16 in any VH structural domain of one group of LCDR also be various embodiment of the present invention.
The VH structural domain usually and the VL structural domain match to provide antibody antigen-binding site, although as discussed further below, VH or VL structural domain can be used for separately conjugated antigen.In one embodiment, antibody 1 VH structural domain and the pairing of antibody 1 VL structural domain, thus form the antibody antigen-binding site that comprises simultaneously antibody 1 VH and VL structural domain.The similar embodiment of other VH described herein and VL structural domain is provided.In other embodiments, the pairing of the VL structural domain beyond antibody 1 VH and antibody 1 VL.Well known light chain mixes.The present invention also provides the similar embodiment of other VH described herein and VL structural domain.Therefore, any VH can match with any VL among parental generation or the antibody 2-16 among parental generation or the antibody 2-16.
An aspect of of the present present invention is the separation antibody molecule that comprises VH structural domain and VL structural domain, and wherein VH structural domain aminoacid sequence is shown in SEQ ID NO:62, and VL structural domain aminoacid sequence is shown in SEQ IDNO:67 or SEQ ID NO:176.
Binding members can comprise among parental generation antibody or the antibody 2-16 any one group of H and/or L CDR, wherein contains 10 or 9 or still less in disclosed H and/or L CDR group, for example 1,2,3,4 or 5 replacement.For example, binding members of the present invention can comprise the H of antibody 7 and/or L CDR group, wherein can comprise 10 or still less, for example 0,1 or 2 replacement.Might any residue in the CDR group be replaced, can be in CDR1, CDR2 and/or CDR3.
Replacement can be in CDR3, and for example any position in antibody 2-16 is shown in table 21.Therefore, described one or more replacement can comprise one or more replacements at following residue place:
Kabat residue 98 among the HCDR3,100A, 101 or 102; Or
Kabat residue 90,92,93,94 among the LCDR3,95 or 96.
Therefore, for example CDR3 can be parental generation (antibody 1) LCDR3 that has replacement at Kabat residue 93 and/or 94 places.
It locates to have described the example that replaces among the parental generation CDR this paper.Described replacement can comprise the one or more replacements shown in the table 21.
Binding members of the present invention can comprise following HCDR1, HCDR2 and/or HCDR3:
HCDR1, wherein Kabat residue 31 is Ser, Ala, Gly, Thr or Cys, for example Ser, and/or Kabat residue 32 is Tyr;
HCDR2, wherein Kabat residue 58 is Tyr or Phe, for example Tyr;
HCDR3, wherein
Kabat residue 96 is hydrophobic residue, namely contains the amino-acid residue of non-polar sidechain, for example Leu, Ile, Val, Ala or Phe, and Leu for example,
Kabat residue 97 is hydrophobic residue, for example Ile, Leu, Val, Ala or Phe, for example Ile and/or
Kabat residue 98 is ring-type residues, and namely side chain comprises the amino-acid residue of circular part.For example, Kabat residue 98 can be His, Trp or Phe, and for example His or Trp are such as His.
Shown in embodiment 7.4, can be in conjunction with IL-17A at the residue of these HCDR positions.
Binding members of the present invention can comprise following LCDR1 and/or LCDR3:
LCDR1, wherein Kabat residue 31 is Tyr or Phe, for example Tyr, and/or Kabat residue 32 is Tyr or Phe, for example Tyr;
LCDR3, wherein Kabat residue 91 is that Tyr and/or Kabat residue 93 are Pro, oxyproline, His, methylhistidine or Asp, for example Pro or His are such as Pro.
Kabat residue 29 among the LCDR1 is optional to be Ala, and/or the Kabat residue among the LCDR1 30 optional be Asn.
Binding members of the present invention can be chosen wantonly and comprise LCDR2, and wherein Kabat residue 53 is to contain the not amino-acid residue of charged polar side chain, for example Gln.
Shown in embodiment 7.4, the residue of these LCDR positions can be in conjunction with IL-17A.
For example, binding members of the present invention can comprise one group of CDR:HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, comprises 7 or more, and for example 8,9 or whole following residues:
Ser, Ala, Gly, Thr or the Cys at Kabat residue 31 places of-HCDR1, for example Ser;
The Tyr at Kabat residue 32 places of-HCDR1;
Tyr or the Phe at Kabat residue 58 places of-HCDR2, for example Tyr;
The hydrophobic residue at-Kabat residue 96 places, for example Leu, Ile, Val, Ala or Phe are such as Leu;
The hydrophobic residue at Kabat residue 97 places of-HCDR3, for example Ile, Leu, Val, Ala or Phe are such as Ile;
The ring-type residue at Kabat residue 98 places of-HCDR3, for example His, Trp or Phe are such as His or Trp, such as His;
Tyr or the Phe at Kabat residue 31 places of-LCDR1, for example Tyr;
Tyr or the Phe at Kabat residue 32 places of-LCDR1, for example Ty;
The Tyr at Kabat residue 91 places of-LCDR3; With
Pro, oxyproline, His or the 3-Methyl histidine at Kabat residue 93 places of-LCDR3, for example Pro or His are such as Pro.
Binding members can comprise one group of CDR, wherein:
The Kabat residue 31 of HCDR1 is Ser;
The Kabat residue 32 of HCDR1 is Tyr;
The Kabat residue 58 of HCDR2 is Tyr;
The Kabat residue 96 of HCDR3 is Leu
The Kabat residue 97 of HCDR3 is Ile;
The Kabat residue 98 of HCDR3 is His;
The Kabat residue 31 of LCDR1 is Tyr;
The Kabat residue 32 of LCDR1 is Tyr;
The Kabat residue 91 of LCDR3 is Tyr; With
The Kabat residue 93 of LCDR3 is Pro.
In binding members of the present invention:
HCDR1 can have 5 amino acid longs, and 31-35 consists of by the Kabat residue;
HCDR2 can have 17 amino acid longs, and 50-65 consists of by the Kabat residue;
HCDR3 can have 9 amino acid longs, and 95-102 consists of by the Kabat residue;
LCDR1 can have 13 amino acid longs, and 24-34 consists of by the Kabat residue;
LCDR2 can have 7 amino acid longs, and 50-56 consists of by the Kabat residue;
And/or
LCDR3 can have 9 amino acid longs, and 89-97 consists of by the Kabat residue.
The illustrating of Kabat numbering of one group of HCDR and LCDR sees Table 21, and wherein HCDR1 is that Kabat residue 31-35, HCDR2 are that Kabat residue 50-65, HCDR3 are that Kabat residue 95-102, LCDR1 are that Kabat residue 24-34, LCDR2 are that Kabat residue 50-56 and LCDR3 are Kabat residue 89-97.
Binding members can be included in has one or more CDR in the antibody framework, for example the antibody molecule of one group of CDR.For example, one or more CDR or one group of CDR grafting of antibody can be entered framework (for example, people's framework) so that antibody molecule to be provided.It is the constant gene segment C sequence that framework region can comprise ethnic group.Therefore, can be with framework kind systemization (germlined), thus the one or more residues in the framework are changed to be that the residue at the framework medium-priced place of putting is complementary to most of similar ethnic groups.The technician can Selective sequence is section near the kind of the antibody framework sequence before the kind systemization, and in test described herein the avidity of this antibody of test or active in to confirm that kind of a systemization does not obviously reduce antigen combination or effectiveness.The known ethnic group of those skilled in the art is the constant gene segment C sequence, can be by for example Vbase compiling (software) access.
In one embodiment, binding members of the present invention is to have to comprise the VH structural domain that ethnic group is one group of HCDR in the framework, for example the isolating human antibodies molecule of VH3-23.Therefore, VH structural domain framework region FR1, FR2 and/or FR3 can comprise the framework region that ethnic group is constant gene segment C VH3-23.FR4 can comprise the framework region that ethnic group is j section JH1, JH4 or JH5 (these j sections have identical aminoacid sequence), perhaps can comprise the framework region that ethnic group is j section JH3.The aminoacid sequence of VH FR1 can be SEQ ID NO:189.The aminoacid sequence of VH FR2 can be SEQ ID NO:190.The aminoacid sequence of VH FR3 can be SEQ ID NO:191.The aminoacid sequence of VH FR4 can be SEQ ID NO:192.Binding members also has the VL structural domain usually, and it for example comprises that ethnic group is framework, such as one group of LCDR among the V λ 6a.Therefore, VL structural domain framework region FR1, FR2 and/or FR3 can comprise the framework region that ethnic group is constant gene segment C V λ 6a.FR4 can comprise the framework region that ethnic group is j section JL2 or JL3 (these j sections have identical aminoacid sequence).The aminoacid sequence of VL FR1 can be SEQ ID NO:193.The aminoacid sequence of VL FR2 can be SEQ ID NO:194.The aminoacid sequence of VL FR3 can be SEQ ID NO:195.The aminoacid sequence of VL FR4 can be SEQ ID NO:196.Planting VH or the VL structural domain of systemization can locate kind of being or not plant systemization at one or more fine setting residues place at one or more fine setting residues (Vernier residue), but does not usually plant system at one or more fine setting residues place.
Antibody molecule of the present invention or VH structural domain can comprise VH FR1, and wherein Kabat residue 28 is Thr or Ser, and for example Thr and/or Kabat residue 30 are Ser or Thr, for example Ser.
Antibody molecule of the present invention or VL structural domain can comprise VL FR2, and wherein Kabat residue 49 is hydrophobic residue, and for example Phe, Tyr or His are such as Phe.
Shown in embodiment 7.4, the Phe49 in the Thr28 in the VH structural domain framework and Ser30 and the VL structural domain framework can be in conjunction with IL-17A.
Antibody molecule of the present invention or VH structural domain can comprise lower group of heavy chain framework region:
FR1 SEQ ID NO：189；
FR2 SEQ ID NO：190；
FR3 SEQ ID NO：191；
FR4 SEQ ID NO：192；
Perhaps, the described one group of heavy chain framework region that comprises contains 1,2,3,4 or 5 amino acid change, for example replaces.
Antibody molecule of the present invention or VL structural domain can comprise lower group of light chain framework region:
FR1 SEQ ID NO：193；
FR2 SEQ ID NO：194；
FR3 SEQ ID NO：195；
FR4 SEQ ID NO：196；
Perhaps, the described one group of light chain framework region that comprises can contain 1,2,3,4 or 5 amino acid change, for example replaces.
Amino acid change can be to replace, insert or disappearance.Choose wantonly and do not make change at the Kabat residue Ser30 of the Kabat of VH FR1 residue Thr28, VH FR1 and/or the Kabat residue Phe49 place of VL FR2.
For example, antibody molecule of the present invention can comprise one group of heavy chain and light chain framework region, wherein:
Heavy chain FR1 is SEQ ID NO:189;
Heavy chain FR2 is SEQ ID NO:190;
Heavy chain FR3 is SEQ ID NO:191;
Heavy chain FR4 is SEQ ID NO:192;
Light chain FR1 is SEQ ID NO:193;
Light chain FR2 is SEQ ID NO:194;
Light chain FR3 is SEQ ID NO:195;
Light chain FR4 is SEQ ID NO:196;
Perhaps, the described one group of heavy chain that comprises and light chain framework region can contain 10 or still less, for example 5 or amino acid change still less for example replace.For example, in described one group of heavy chain and light chain framework region, 1 or 2 aminoacid replacement can be arranged.
Compare with kind of being antibody, the antibody of not planting systemization has identical CDR, but framework is different.
In antibody sequence shown in this article, with antibody 2,3,4,5,6,7,13,14 and 15 VH structural domain kind systemization, antibody 1,8,9,10,11,12 and 16 VH structural domain are not planted being, with the VL structural domain kind systemization of antibody 2 and 7, antibody 1,3,4,5,6,8,9,10,11,12,13,14,15 and 16 VL structural domain are not planted being.
Binding members of the present invention can be competed in conjunction with IL-17 (all in conjunction with IL-17) with any binding members, comprises binding members, VH and/or VL structural domain, CDR that this paper discloses, for example HCDR3 and/or CDR group.Be not difficult competition between external check binding members, for example adopt ELISA and/or mark (can in detection in the presence of one or more other unlabelled binding members) for a kind of binding members with specific reporter molecule, thereby can identify the binding members in conjunction with identical epi-position or overlapping epi-position.For example, can adopt ELISA to measure competition, wherein IL-17 is fixed in flat board, and the first binding members of mark is added this flat board together with one or more unlabelled other binding members.The signal that binding members by mark sends reduces to be observed and can exist with the unmarked binding members of the binding members competition of mark.Those of ordinary skills are not difficult to know these methods, and this paper describes these methods in detail.In one embodiment, adopt epi-position competition experiments check competition combination as herein described.Binding members of the present invention can comprise the antibody antigen-binding site with antibody molecule competition, for example particularly comprises parental generation antibody or in conjunction with any VH and/or VL structural domain, CDR, for example antibody molecule of HCDR3 or CDR group among the antibody 1-16 of IL-17.Aspects of the present invention provide and any binding members described herein competition in conjunction with the binding members of IL-17, for example with parental generation antibody or antibody 2-16 in any competition, for example scFV or IgG1 form.Compete any one or more structure and/or the functional performance that can have the binding members of the present invention of this paper disclosure in conjunction with the binding members of IL-17 with any binding members described herein.
In other side, the invention provides the isolating nucleic acid that comprises code book invention binding members, VH structural domain and/or VL structural domain, and the method for preparing binding members of the present invention, VH structural domain and/or VL structural domain, be included in to cause and express described nucleic acid under the condition that produces described binding members, VH structural domain and/or VL structural domain and reclaim it.
The present invention provides the coding VH CDR that this paper discloses or the nucleic acid of VL CDR sequence on the other hand, normally separates.
The host cell that contains nucleic acid of the present invention or transform with nucleic acid of the present invention is provided on the other hand.
Other side of the present invention provides the composition that contains binding members of the present invention, and they suppress and/or in and application in the method for IL-17, comprise the method for the treatment of human or animal body.
Binding members of the present invention can be used for treating or diagnosing the method for human or animal body, for example treats (can comprise prophylactic treatment) disease of people patient or the method for illness, and the method comprises the binding members of the present invention that gives described patient's significant quantity.Can comprise any illness that IL-17 works therein according to the illness of the present invention's treatment, such as its place's detailed description of this paper.
Hereinafter be described in further detail these and other aspect of the present invention.
Term
Should point out herein, this paper use " and/or " part should be understood to each that specifically discloses two kinds of described features or component, comprises or do not comprise another kind.For example, " A and/or B " should be understood to specifically disclose (i) A, (ii) B and (iii) among A and the B each, just as listing specially in this article separately.
IL-17
IL-17 or IL-17A are interleukin-17s.Unless statement is arranged in addition, addresses IL-17 and be often referred to human il-17 A.
The IL-17A of expression in vivo has 23 terminal signal peptides of amino acid whose N-, and it produces ripe IL-17A through cutting.The sequence of wild-type human il-17 A is SEQ ID NO:198.
The amino-acid residue of addressing among the IL-17 is normally numbered according to the residue of mature sequence, and does not comprise signal peptide.Unless statement is arranged in addition, the residue of human il-17 numbering refers to mature sequence SEQ ID NO:198 herein.The residue 1 of ripe IL-17A is Gly, and it is positioned at 24 of full-length polypeptide.
The sequence of human il-17 is with accession number Q16552 preservation (Swiss-Prot), and its demonstration comprises the total length precursor I L-17A of signal peptide.
The sequence of stump-tailed macaque IL-17A is shown as SEQ ID NO:162, and it is encoded by SEQ ID NO:161.
State such as its place of this paper, IL-17A can be recombinant chou and/or can be glycosylation or nonglycosylated.The natural expression of glycosylation form that IL-17A connects with N-in vivo.Glycosylation IL-17A also can be at recombination system, for example HEK EBNA cells.IL-17A can be with non-glycosylated formal representation in Bacillus coli cells.
Binding members
This term description a member in a pair of molecule that is bonded to each other.Can natural generation or wholly or in part synthetic generation in conjunction with right member.The right member of molecule has certain area or cavity on its surface, thereby can be combined to another member's particular space and polar structure with this molecule, and is therefore complementary with it.Example in conjunction with right type is Ag-Ab, vitamin H and avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate.The present invention relates to the reaction of Ag-Ab type.
Binding members generally includes the molecule with antigen binding site.For example, binding members can be antibody molecule or the non-antibody albumen that contains antigen binding site.
Can be by arrange CDR (Haan and Maggos, 2004 at the protein scaffolds such as the non-antibody such as fibronectin or cytochrome B; Koide, 1998; Nygren, 1997), or by making the amino-acid residue randomization or the sudden change that encircle in the albumen support that antigen binding site is provided, thereby can give the binding specificity to required target.Nygren etc., (1997) have summed up the support that is used for the novel binding site of engineered protein.The albumen support of antibody analog is disclosed in the WO/0034784 that includes in full by reference this paper in, and wherein the contriver has described the protein (antibody analog) that comprises the fibronectin III type structural domain with at least one randomization ring.Can provide by any structural domain member of immunoglobulin gene superfamily and want grafting to enter one or more CDR, for example the suitable holder of one group of HCDR or HCDR and/or LCDR3.Described support can be people or non-human protein's matter.One of advantage of non-human protein's matter support is that it can provide antigen binding site in less than some antibody molecules at least and/or the support molecule that is easier to operate.The volume I of binding members is given useful physiology characteristic, for example can enter cell, penetrate deeper into tissue or with other structure in target reaction, or be incorporated in the protein cavity of target antigen.Wess, 2004 have summed up the application of antigen binding site in the non-antibody protein scaffolds.Protein has stable skeleton and one or more variable loop usually, thereby the aminoacid sequence of wherein said one or more rings produces the antigen binding site of being combined with target antigen through special or random mutation.These protein comprise the IgG-binding domains of staphylococcal protein A, Transferrins,iron complexes, tetranectin, fibronectin (for example, the 10th III type fibronectin structural domain) and NGAL.Other method comprises synthetic " microbody " of Xi Laike limited-liability company (SelecoreGmbH), and it is take plant cyclase protein (cyclotide) (small protein with intramolecular disulfide bond) as the basis.
Except antibody sequence and/or antigen binding site, binding members of the present invention can comprise other amino acid, for example forms peptide or polypeptide, such as fold domain, or gives this molecule except in conjunction with the another kind of functional character the ability of antigen.Binding members portability detectable of the present invention, perhaps can with toxin or targeting moiety or enzyme coupling (for example, by peptide bond or joint).For example, binding members can comprise catalytic site (for example, in the enzymatic structure territory) and antigen binding site, and wherein said antigen binding site is combined with antigen, so the catalytic site of targeting antigen.Catalytic site can, for example suppress the biological function of antigen by cutting.
Although know non-antibody support portability CDR, but carry CDR, normally heavy chain of antibody or sequence of light chain or its substantial part of the structure of CDR3 or one group of CDR of the present invention for example, wherein CDR or CDR group is positioned at natural generation VH and the CDR of VL antibody variable domains or the corresponding position of CDR group of the immunoglobulin gene coding of rearrangement.Can be with reference to utilizing at present the Internet can obtain the Kabat 1987 of (immuno.bme.nwu.edu or adopt any search engine retrieving " Kabat ") and structure and the location that upgraded version is measured the immunoglobulin variable territory thereof.
CDR zone or CDR show the heavy chain of immunoglobulin (Ig) of Kabat etc. (Kabat 1991a, and version subsequently) definition and the hypervariable region of light chain.Antibody contains 3 heavy chain CDR and 3 light chain CDR usually.This paper utilize term CDR represent in (playing by ear) these zones one or several or even all, these zones are contained and are responsible for by avidity so that most of amino-acid residues that antibody is combined with antigen or the epi-position of its identification.
In 6 short CDR sequences, the variable volume of the 3rd CDR (HCDR3) of heavy chain large (mainly being to differ greatly because produce its gene rearrangement mechanism).It can be as short as 2 amino acid, although the longest known volume is 26.On function, HCDR3 plays partial action (Segal1974 in determining antibodies specific; Amit 1986; Chothia 1987; Chothia 1989; Caton 1990; Sharon 1990a; Sharon 1990b; Kabat etc., 1991b).
HCDR1 can be 5 amino acid longs, and 31-35 consists of by the Kabat residue.
HCDR2 can be 17 amino acid longs, and 50-65 consists of by the Kabat residue.
HCDR3 can be 9 amino acid longs, and 95-102 consists of by the Kabat residue.
LCDR1 can be 13 amino acid longs, and 24-34 consists of by the Kabat residue.
LCDR2 can be 7 amino acid longs, and 50-56 consists of by the Kabat residue.
LCDR3 can be 9 amino acid longs, by Kabat residue 89-97.
Antibody molecule
This term description the natural or partially or completely synthetic immunoglobulin (Ig) that produces no matter.Any polypeptide or the protein that comprises antibody antigen-binding site also contained in this term.Must know in this article, the present invention does not relate to the antibody of natural form, be that they are not in its natural surroundings, but they can purifiedly separate or acquisition from natural origin, perhaps can obtain by genetic recombination or chemosynthesis, they can contain alpha-non-natural amino acid subsequently, hereinafter will further describe.The antibody fragment that contains antibody antigen-binding site includes but not limited to: such as Fab, Fab ', Fab '-SH, scFv, Fv, dAb, Fd equimolecular; And double antibody (diabody).
Antibody molecule of the present invention can be IgG, for example IgG1.
May utilize monoclonal antibody or other antibody and adopt the technology such as recombinant DNA technology to produce other antibody or mosaic type molecule in conjunction with target antigen.These technology can comprise introduces the coding immune globulin variable region, or the CDR of antibody, or the constant region of different immunoglobulin (Ig)s or constant region add the DNA of framework region.Referring to, for example EP-A-184187, GB 2188638A or EP-A-239400, and a large amount of follow-up documents.Can make genetic mutation or other change to the hybridoma or other cell that produce antibody, thereby can change or not change the binding specificity of the antibody that produces.
Have required specificity and/or can be combined with antigen because modified antibodies in many ways, term " antibody molecule " should be understood to contain, have any binding members or the material of antibody antigen-binding site.Therefore, antibody fragment and derivative contained in this term, comprises any polypeptide that contains antibody antigen-binding site, and no matter it is natural or synthetic wholly or in part.Therefore comprise the mosaic type molecule that comprises antibody antigen-binding site and merge with another polypeptide (for example, derived from another species or belong to another Antibody types or subclass), or its Equivalent.EP-A-0120694 and EP-A-0125023 and a large amount of follow-up document description clone and the expression of mosaic type antibody.
Other available in antibody engineering field technology can be separated people and humanized antibody.For example, can be such as preparation people hybridoma as described in Kontermann and the Dubel (2001).For example following many publications are described the another kind of prior art-phage display that produces binding members: WO92/01047 (hereinafter further discussing) and US Patent No. 5969108, US5565332, US5733743, US5858657, US5871907, US5872215, US5885793, US5962255, US6140471, US6172197, US6225447, US6291650, US6492160, US6521404, Kontermann and Dubel (2001) in detail.Can utilize the transgenic mice isolating human antibodies, this mouse small mouse antibody gene is inactivated and the functional human immunoglobulin gene that replaces with, but other component of mouse immune system is kept intact (Mendez 1997).
Can be by oligonucleotide synthetic and in suitable expression vector assembling produce gene, produce synthetic antibody molecule by expressing gene, such as Knappik etc., (2000) or Krebs etc., (2001) are described.
Proved that now the fragment of complete antibody can carry out the function of conjugated antigen.The example of binding fragment is the Fab fragment that (i) is made of VL, VH, CL and CH1 structural domain; (ii) the Fd fragment that is consisted of by VH and CH1 structural domain; (iii) the Fv fragment that is consisted of by VL and the VH structural domain of monospecific antibody; (iv) (Ward 1989 for the dAb fragment that is made of VH or VL structural domain; McCafferty 1990; Holt 2003); The CDR zone of (v) separating; (vi) F (ab ') 2 fragments, it is a kind of divalence fragment that comprises the Fab fragment of two connections; (vii) scFv molecule (scFv), wherein the VH structural domain links to each other by peptide linker with the VL structural domain, (Bird 1988 to form antigen binding site thereby can connect two structural domains; Huston1988); (viii) dual specific scFv dimer (PCT/US92/09965) and " double antibody " that (ix) make up by gene fusion, multivalence or polyspecific fragment (WO94/13804; Holliger 1993a).Can stablize Fv, scFv or double antibody molecule (Reiter1996) by comprising the disulfide linkage that connects VH and VL structural domain.Also can prepare the corpusculum (minibody) (Hu 1996) that comprises the scFv that links to each other with the CH3 structural domain.Other example of binding fragment is Fab ', the difference of itself and Fab fragment is to add a little residue at the C-terminal of heavy chain CH1 structural domain, the one or more halfcystines that comprise the antibody hinge region, and Fab '-SH, it is the Fab ' fragment of the many free sulfhydryl groups of cysteine residues of constant region.
Can by such as enzyme (such as stomach en-or papoid) digestion and/or chemical reduction with methods such as cutting disulfide linkage, begin to obtain antibody fragment of the present invention from any antibody molecule as herein described, for example comprise the antibody molecule of the CDR of any among VH and/or VL structural domain or the antibody 1-16.In another kind of mode, can be by technology such as genetic recombination well known to those skilled in the art, perhaps by, automatic peptide synthesizer for example, those instruments that provide such as companies such as Applied Biosystems, Inc. (Applied Biosystems) are synthetic or synthetic and express and obtain antibody fragment of the present invention by nucleic acid by peptide.
Functional antibodies fragment of the present invention comprises by chemically modified, particularly by PEGization, or increased any functional fragment of transformation period by mixing liposome.
DAb (domain antibodies) is the minor comonomer Fab of antibody, i.e. the variable region of heavy chain of antibody or light chain (Holt 2003).VH dAb natural generation in camelidae animal (for example, camel, yamma), and can be by using the hunchbacked class animal of target antigen immunity, the dAb gene that separates antigen-specific b cells and each B cell of Direct Cloning produces.Also can in cell culture, produce dAb.Volume is little, favorable solubility and temperature stability be so that they are particularly useful on physiology, thereby is suitable for selecting and affinity maturation.Binding members of the present invention can be to comprise VH basically as described herein or VL structural domain or comprise basically the VH of one group of CDR as described herein or the dAb of VL structural domain.
The feature of the VH of phrase used herein " basically described " expression binding members described herein or the relevant CDR of VL structural domain is identical with the specific region of sequence described herein or highly similar.As described herein, be included in CDR and/or VH or the VL structural domain about the phrase " highly similar " of one or more variable domains and can carry out about 5 of 1-, for example 1-4 comprises 1-3, or 1 or 2 or 3 or 4 aminoacid replacement.
Dual specific or bifunctional antibody formation are combined in two different variable regions with the s-generation monoclonal antibody in a part (Holliger 1999).Proved that now they can be used for diagnostic field and treatment field because of several molecules that can raise new effector function or target tumor cell surface.If utilize bi-specific antibody, these antibody can be with ordinary method (Holliger 1993b), and for example chemical process prepares or from the conventional bi-specific antibody that hybridoma prepares, perhaps can be any of above-mentioned bispecific antibody fragment.These antibody can pass through chemical process, and (Glennie 1987; Repp 1995) or the somatocyte method (Staerz 1986; Suresh 1986) obtain, but also can carry out different dimerization to force by the genetic modification technology, thereby promote to look for the purge process (Merchand 1998) of antibody.The example of bi-specific antibody comprises BiTE
TMThose antibody that technology obtains wherein can utilize the binding domains of two kinds of different antibody of specificity and pass through short snappiness peptide directly to link to each other.This antibody is at short two kinds of antibody of Single polypeptide chain combination.Can only utilize the variable region to make up and not contain double antibody and the scFv in Fc district, thereby may reduce the effect of anti-idiotype reaction.
Bi-specific antibody can be built into complete IgG, dual specific Fab ' 2, Fab ' PEG, double antibody or dual specific scFv.In addition, can adopt ordinary method known in the art to connect two bi-specific antibodies to form tetravalent antibody.
Opposite with complete bi-specific antibody, dual specific two antibody may be also particularly useful, because they are not difficult to make up and at expression in escherichia coli.Adopt phage display (WO94/13804) to be not difficult from the library, to select suitable two antibody (with many other polypeptide, for example antibody fragment) of binding specificity.If an arm of double antibody is kept constant, for example to the specificity of IL-17, then can prepare the different library of another arm and select the suitable antibody of specificity.Can as described in Ridgeway 1996, prepare complete bi-specific antibody by replacing engineered method.
This area can adopt the whole bag of tricks acquisition for the antibody of IL-17.These antibody can be monoclonal antibody, particularly people, Muridae, mosaic type or humanization source, and these antibody can obtain by standard method well known to those skilled in the art.
Just prepare monoclonal antibody or their function fragment, mouse source particularly, usually may represent the special technology of describing of handbook " Antibodies (antibody) " (Harlow and Lane 1988) or such as Kohler and Milstein, the 1975 described technology that prepare from hybridoma.
Monoclonal antibody can from, for example obtain with IL-17 or the zooblast of one of their fragments immunity that contains the epi-position of described monoclonal antibody identification.Especially can be according to ordinary method, the genetic recombination that the nucleotide sequence of the cDNA sequence by certainly containing coding IL-17 or its fragment begins, the peptide that begins by self-contained aminoacid sequence in the peptide sequence of IL-17 and/or its fragment synthesizes to prepare one of IL-17 or its fragment.Monoclonal antibody can be utilized, and affinity column purifying has for example been fixed one of the IL-17 of the epi-position that contains described monoclonal antibody identification or its fragment on the described affinity column.More particularly, can pass through A albumen and/or G protein chromatographic, then carry out or do not carry out ion exchange chromatography to remove residual protein pollutent and DNA and LPS in self, carry out again or do not carry out the sepharose exclusion chromatography and come monoclonal antibody purification with the potential aggregate of removing because existing dimer or other polymer to cause.In one embodiment, can adopt simultaneously or adopt continuously all these technology.
Antigen binding site
This term description in the molecule in conjunction with all or part of of target antigen and complementary part with it.In antibody molecule, this term represents antibody antigen-binding site, comprises in the antibody in conjunction with all or part of of target antigen and complementary part with it.If antigen is larger, antibody can be only in conjunction with the specific part of this antigen, and this part is called epi-position.Can provide antibody antigen-binding site by one or more antibody variable domains.Antibody antigen-binding site can comprise antibody chain variable region (VL) and antibody heavy chain variable region (VH).
Separate
This term represents binding members of the present invention, or the nucleic acid of this binding members of encoding conforms with state of the present invention usually.Therefore, the binding members of the present invention that provides, VH and/or VL structural domain and coding nucleic acid molecule and carrier can be for example separate from its natural surroundings and/or purifying, basically form pure or homogeneous, perhaps in the situation of nucleic acid, has the sequence of polypeptide of required function with nucleic acid or the gene of other sequence of external source for not containing or being substantially free of coding.The member who separates does not contain with the nucleic acid that separates or is substantially free of natural coupled material, for example implements at its natural surroundings or in external or body to prepare other polypeptide or the nucleic acid of finding in environment (for example cell culture fluid) at it in the recombinant DNA technology.Available thinner or adjuvant are prepared all members and nucleic acid, but for actual purpose be still separation-for example, if be used for the coated used microtiter plate of immunoassay, usually these members are mixed with gelatin or other carrier, when perhaps being used for diagnosis or treatment, with itself and pharmaceutically acceptable carrier or mixing diluents.Binding members can be natively or the system's glycosylation by allos eukaryotic cell (for example, CHO or NS0 (ECACC 85110503)), and perhaps they can be that (for example, if express in prokaryotic cell prokaryocyte) is nonglycosylated.
The heterogeneous goods that comprise the IL-17 antibody molecule also consist of a part of the present invention.For example, these goods can be mixtures of antibodies, and described antibody has total length heavy chain, the heavy chain that lacks the terminal Methionin of C-, degree of glycosylation difference and/or has the amino acid of derivatize, and for example the cyclisation of N-terminal glutamate is to form the Pyroglutamic Acid residue.
The accompanying drawing summary
The end-labelled human il-17 A of Fig. 1 .N-aminoacid sequence (SEQ ID NO:197).Underscore has shown the end-labelled residue 1-21 of N-.The residue 92-108 (SEQ ID NO:199) corresponding to the residue 71-87 of ripe human il-17 A in the frame.
Fig. 2. contain the total length precursor human il-17 A aminoacid sequence (SEQ ID NO:200) of the terminal His5 label of C-.Underscore has shown the terminal signal peptide of the N-of 23 amino-acid residues.Corresponding to the adding in the frame sequence SEQ ID NO:199 of the residue 71-87 of ripe human il-17 A, the sudden change residue of wild-type human il-17 A (SEQ ID NO:198) shows with boldface type and underscore.The ripe mutant human IL-17A that does not contain N-terminal peptide and His mark is SEQ ID NO:201.
Describe in detail
As mentioned above, binding members of the present invention can regulate and may in and the biologic activity of IL-17.As described herein, can optimize the neutralization of IL-17 binding members of the present invention and render a service.Effectiveness optimization generally includes sequence (the normally variable domain sequence of the antibody) sudden change of the binding members that makes selection to produce the binding members library, then testing effect and the more potent binding members of selection.Therefore, the effectiveness of " effectiveness advantage " binding members of selection may be higher than the binding members that produces the library.Yet, also can obtain the efficient binding members without optimizing, for example can be from initial screening, the test of biological example chemical neutralization directly obtains the efficient binding members." render a service and optimize " binding members refer in and the binding members optimized of the effectiveness of the given activity of IL-17 or downstream function.Its place's more detailed description of this paper test and render a service.The invention provides and render a service the binding members of optimizing and do not optimize, and render a service the method for optimizing selected binding members.Therefore, the invention enables the technician can produce efficient binding members.
Render a service higher binding members although can adopt render a service to optimize from given binding members, to produce, should note even can render a service optimizing and obtaining efficient binding members.
On the other hand, the invention provides the method for one or more binding members of acquisition energy conjugated antigen, the method comprises makes binding members of the present invention library contact with described antigen, and selecting can be in conjunction with one or more binding members of described antigen in this library.
This library can be illustrated on particle or the molecular complex; reproducible hereditary wrapping body (replicable genetic package) for example; such as yeast, bacterium or bacteriophage (for example; T7) particle, virus, cell or covalency, rrna or other external display systems, each particle or molecular complex contain the nucleic acid that coding is showed antibody VH variable domain thereon and chosen the VL structural domain (if present) of showing wantonly.Following document description phage display: WO92/01047 and for example, US Patent No. 5969108, US5565332, US5733743, US5858657, US5871907, US5872215, US5885793, US5962255, US6140471, US6172197, US6225447, US6291650, US6492160 and US6521404, each piece document mode is by reference included this paper in full in.
Select can conjugated antigen and be illustrated in bacteriophage or other library particle or molecular complex on binding members after, can obtain nucleic acid from the bacteriophage of the binding members of showing described selection or other particle or molecular complex.This nucleic acid can be used for subsequently containing the nucleotide sequence that obtains nucleic acid from the bacteriophage of the binding members of showing described selection or other particle or molecular complex by expression and produces binding members or antibody VH or VL variable domain.
The antibody VH variable domain of aminoacid sequence of antibody VH variable domain that contains the binding members of described selection can provide by unpack format, for example comprises the binding members of this VH structural domain.
Further test in conjunction with the antibody of IL-17 with, for example parental generation antibody molecule or antibody molecule 1-16 (for example, scFv form and/or IgG form are such as IgG1) competition is in conjunction with the ability of IL-17.In and the ability of IL-17 can state test such as its place of this paper.
Binding members of the present invention is identical or better in conjunction with one of the avidity of IL-17 and antibody 1-16 (for example, scFv or IgG1 form).
Identical or better with one of the effectiveness of the biologic activity of IL-17 and antibody 1-16 (for example, scFv or IgG1 form) in the binding members of the present invention.
Can render a service with neutralization at the binding affinity of more different binding members under the conditions suitable.
Can prepare the variant of antibody molecule disclosed herein and be used for the present invention.Guidance according to calculational chemistry is applied to structure/characteristic-activity relationship (Wold 1984) with the multivariate data analysis technology, can adopt the mathematical technique of knowing, and for example (Norman 1998 for statistical regression, pattern recognition and classification; Kandel and Backer1995; Krzanowski 2000; Witten and Frank 1999; Denison (volume) 2002; Ghose and Viswanadhan) obtain the quantitative activity-characteristic relation of antibody.The characteristic that can obtain antibody from experience and the theoretical model (for example, the residue that analysis may contact or the physicochemical property of calculating) of antibody sequence, function and three-dimensional structure, these characteristics can be considered separately, also consideration capable of being combined.
Antibody antigen-the binding site that is made of VH structural domain and VL structural domain is formed by 6 polypeptide rings usually: 3 from light chain variable territory (VL), and 3 from heavy chain variable domain (VH).(Chothia 1992 the analysis of the known antibody of atomic structure have been illustrated relation between the three-dimensional structure of sequence and antibody combining site; Al-Lazikani 1997).These concern hint, and the 3rd district (ring) in the VH structural domain, the binding site ring has a small amount of Conformation of the main chain: one of classical architecture.In specific ring, form the classical architecture demonstration by its size and in the critical sites of ring and framework region, exist some residue to determine that (Chothia 1992; Al-Lazikani1997).
Can adopt in the antibody of the known but three-dimensional structure the unknown of this sequence-structural relation research forecasting sequence, for the three-dimensional structure of keeping its CDR ring thereby keep vital those residues of binding specificity.Result by comparison prediction and preamble optimization experiment can support these predictions.In structural approach, adopt any software package that can freely use or buy, for example WAM (Whitelegg and Rees 2000) but production model (Chothia 1986).Then can adopt protein range estimation and analysis software package, for example the possible replacement in position among the Insight II of Acker Sai Leli company (Accelerys, Inc.) or Deep View (Guex and Peitsch 1997) the assessment CDR.Then can utilize this information to carry out may the minimum or favourable replacement on the impact of activity.
This area is usually known to replace required technology in the sequence of CDR, antibody VH or VL structural domain and binding members.Can prepare the variant sequence that contains replacement, test its in conjunction with and/or in and ability and/or any other desired characteristic of IL-17, described replacement may estimate maybe can not estimate that the impact on activity is minimum or favourable.
The present invention can utilize the variable domain amino acid sequence variants of the disclosed especially any VH of sequence such as this paper and VL structural domain, and is as described herein.Concrete variant can comprise one or more aminoacid sequences and change (interpolation of an amino-acid residue, disappearance, replacement and/or insertion), can be less than about 20 changes, less than about 15 changes, less than about 10 changes or less than about 5 changes, it can be 5,4,3,2 or 1.Can in one or more framework regions and/or one or more CDR, make change.Change and usually not cause afunction, the binding members that therefore comprises the aminoacid sequence of change like this can keep in conjunction with and/or in and the ability of IL-17.For example, it can keep quantitative combination and/or the neutralising capacity same with the binding members of not doing to change, and for example records in test described herein.Comprise change like this aminoacid sequence binding members in conjunction with and/or in and the ability of IL-17 may improve.
Change can comprise with non-natural generation or the alternative one or more amino-acid residues of non-standard amino acid, become non-natural to produce or non-standard form one or more Modification of amino acid residues, perhaps one or more non-naturals are produced or the non-standard amino acid insertion sequence.It locates to have described exemplary number and the position that changes in the sequence of the present invention this paper.The amino acid of natural generation comprises 20 kinds of " standard " L-amino acid, differentiates according to their standard one-letter code to be G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E.Non-standard amino acid comprises and can mix any other residue in the polypeptide main chain because of the modification of existing amino-acid residue.Non-standard amino acid can be natural generation or non-natural produce.The non-standard amino acid of several natural generations known in the art, (Voet and Voet 1995) such as 4-Hydroxyproline, 5-OxoPro, 3-Methyl histidine, N-ethanoyl Serine.It is terminal that those amino-acid residues of deriving at its N-alpha position only are positioned at the N-of aminoacid sequence.In the present invention, amino acid is L-amino acid normally, but in some embodiments, also can be D-amino acid.Therefore, change and to comprise the amino acid with the amino acid modified one-tenth of L-D-, or with D-amino acid replacement L-amino acid.Amino acid whosely methylate, acetylize and/or phosphorylation form also be known, in the present invention, amino acid can carry out this modification.
The aminoacid sequence of antibody structure of the present invention territory and binding members can comprise above-mentioned non-natural or non-standard amino acid.In some embodiments, can be between synthesis phase with non-standard amino acid (for example, D-amino acid) mix aminoacid sequence, although in other embodiments, can behind synthetic amino acid array, introduce non-standard amino acid by the standard amino acid of modifying or substitute " original ".
The amino acid that utilizes non-standard and/or non-natural to produce can increase structure and function difference, therefore can increase binding members of the present invention obtain required IL-17 in conjunction with in and the possibility of characteristic.In addition, compare with standard L-amino acid, D-amino acid and analogue have shown better pharmacokinetic profiles overview, are giving animal because have the amino acid whose polypeptide of L-, for example can vivo degradation behind the people.
Can adopt VH and/or the VL gene of the one or more selections of random mutagenesis to suddenly change in whole variable domain, to produce, thereby produce novel VH or the VL district of the CDR of carrying derived sequence of the present invention.Gram etc. (1992) have described this technology, and he adopts fallibility PCR.In some embodiments, in the whole variable region of CDR group, make one or two aminoacid replacement.Adoptable another kind of method is the site-directed mutagenesis in the CDR zone of VH or VL gene.(1996) such as Barbas etc. (1994) and Schier disclose these technology.
All above-mentioned technology known in the art, the technician can adopt these technology, thereby can utilize this area ordinary method that binding members of the present invention is provided.
The present invention provides the method for the antibody antigen-binding site that obtains IL-17 on the other hand, the method comprises by with one or more aminoacid addition, deletion, the aminoacid sequence of VH structural domain provides the VH structural domain shown in replacement or insertion this paper, it is the aminoacid sequence variant of described VH structural domain, the so provided VH structural domain of optional combination and one or more VL structural domain, and test binding members or the antibody antigen-binding site that this VH structural domain or one or more VH/VL make up to identify IL-17, optional have one or more functional performances, for example in and the ability of IL-17 activity.The aminoacid sequence of described VL structural domain is basically as shown here.Can adopt similar approach, wherein one or more sequence variants of VL structural domain disclosed herein and one or more VH domain combination.
As mentioned above, entrained basically cdr amino acid sequence as shown here can be CDR or its substantive part in people's antibody variable domains.Basically HCDR3 sequence as shown here is representing many embodiments of the present invention, and each these sequence of for example carrying can be HCDR3 or its substantive parts in people's heavy chain variable domain.
The used variable domain of the present invention can or be derived from any kind of system or rearrangement people variable domain acquisition, perhaps can be based on the synthetic variable domain of the total or actual sequence of known person variable domain.Variable domain can be derived from the non-human antibody.Can adopt recombinant DNA technology that CDR sequence of the present invention (for example, CDR3) introduce is lacked CDR (for example, CDR3) variable domain storehouse (repertoire).For example, Marks etc. (1992) have described the method that produces the antibody variable domains storehouse, and wherein the total primer of the 3rd framework region of variable domain 5 ' end place or the total primer that adjoins with it and people VH gene is used in conjunction to provide the VH variable domain storehouse that lacks CDR3.How Marks etc. have also described the CDR3 combination with this storehouse and specific antibodies.Adopt similar technology, CDR3 derived sequence of the present invention can be reorganized with the VH that lacks CDR3 or VL structural domain storehouse, and the complete VH of reorganization and VL structural domain and homology VL or VH domain combination are to provide binding members of the present invention.Then can in the appropriate host system, show this storehouse, thereby can select suitable binding members, described host system for example is WO92/01047 or a large amount of follow-up document of including in full by reference this paper in, comprise Kay, the described phage display system of Winter and McCafferty (1996).A storehouse can be by 10
4More than single member's formation, for example at least 10
5, at least 10
6, at least 10
7, at least 10
8, at least 10
9Or at least 10
10Individual member.Other appropriate host system includes but not limited to: yeast displaying, bacterium displaying, T4 displaying, viral displaying, cell display, ribosomal display and covalency are showed.
Stemmer (1994) also discloses similar reorganization or recombinant technology, and he has described the technology relevant with the β-lactamase gene, can adopt the method to produce antibody but observe.
The method of the binding members of preparation IL-17 antigen is provided, and the method comprises:
(a) provide the coding VH initial storehouse of nucleic acid of structural domain, described nucleic acid comprises CDR3 to be substituted or lacks the CDR coding region;
(b) with described nucleic acid library and the basically donor nucleic acid combination of the aminoacid sequence of VH CDR3 as shown here of coding, thereby described donor nucleic acid is inserted the CDR3 zone of nucleic acid library, and then the coding VH nucleic acid product storehouse of structural domain is provided;
(c) nucleic acid in the described product of expression storehouse;
(d) binding members of selection IL-17; With
(e) reclaim described binding members or its coding nucleic acid.
Also can adopt similar approach, wherein VL CDR3 of the present invention makes up with the nucleic acid library of coding VL structural domain, and described nucleic acid comprises CDR3 to be substituted or lacks the CDR3 coding region.
Similarly, one or more or all 3 CDR graftings can be entered VH or VL structural domain storehouse, then screen one or more binding members of IL-17 in this storehouse.
In one embodiment, can utilize one or more among parental generation or antibody 2-16 HCDR1, HCDR2 and the HCDR3, perhaps parental generation or antibody 2-16 HCDR organize, and/or one or more among parental generation or antibody 2-16 LCDR1, LCDR2 and the LCDR3, perhaps parental generation or antibody 2-16 LCDR group.
Similarly, can utilize other VH disclosed herein and VL structural domain, organize CDR and many group HCDR more and/or organize LCDR more.
In one embodiment, the substantive part in immunoglobulin variable territory comprises described at least three CDR districts, and they interleave framework region.This part also can comprise at least about 50% the first or the 4th framework region or the two, and described 50% is the N-end 50% of terminal the 50% and the 4th framework region of C-of the first framework region.The N-end of the substantive part of variable domain or other residue of C-end can be those residues that usually do not link to each other with the variable domain of natural generation.For example, providing recombinant DNA technology to make up binding members of the present invention can cause introducing introducing with the joint that promotes clone or other operation steps coded N-or C-terminal residue.Other operation steps comprises introduces joint connecting variable domain of the present invention and to comprise antibody constant region, other variable domain (for example, in the generation of two antibody) and maybe can detect/the other oroteins sequence of functional label thing, describes in detail such as its place of this paper.
Although aspect more of the present invention, binding members comprises a pair of VH and VL structural domain, consist of other side of the present invention based on the single binding domains of VH or VL structural domain sequence.Known single immunoglobulin domains, particularly VH structural domain can be with ad hoc fashion in conjunction with target antigens.For example, referring to the discussion of above dAb.
In the situation of each single binding domains, available these structural domains screening can form and the IL-17 energy in conjunction with the complementary structure territory of bidimensional binding members.Can adopt the dual assembled scheme of the disclosed so-called classification of the WO92/01047 that includes in full with way of reference, implement by the phage display screening method, each colony that wherein contains H or L chain clone is used for the clone's of infection another chain of coding (L or H) complete library, according to display technique of bacteriophage, the double-stranded binding members that those choices of technology of for example describing in this reference obtain.Marks 1992 has also disclosed this technology.
Binding members of the present invention also can comprise antibody constant region or its all part, for example people's antibody constant region or its all part.For example, can with the VL structural domain its C-terminal with contain people C κ or C λ chain, for example the antibody light chain constant region of C λ chain links to each other.Similarly, binding members based on the VH structural domain can be at its C-end and derived from any antibody isotype, for example all or part of (for example, the CH1 structural domain) of the heavy chain immunoglobulin of IgG, IgA, IgE and IgM and any isotype subclass, particularly IgG1 and IgG4 links to each other.IgG1 is preferred because of its effector function and convenient operation.Any synthesize or other constant region variant also can be used in the embodiment of the present invention that has these characteristics and stablize the variable region.
The available detection or functional label substance markers binding members of the present invention.Marker can be can produce or through inducing any molecule that produces signal, include but not limited to: fluorescent agent, radio-labeling, enzyme, chemoluminescence agent or photosensitizers.Therefore, can detect and/or measure in conjunction with situation by detecting fluorescence or luminous, radioactivity, enzymic activity or absorbancy.
Suitable marker comprises (exemplary and non-limiting) enzyme, for example alkaline phosphatase, glucose-6-phosphate dehydrogenase (G6PD) (" G6PDH ") and horseradish peroxidase; Dyestuff; Fluorescent agent, for example fluorescein, rhodamine compound, phycoerythrin, Phycocyanins, C-, allophycocyanin, o-phthalaldehyde(OPA), fluorescamine, fluorophore, for example lanthanide series compound and sequestrant (the biological international corporation (PerkinElmer and Cis Biointernational) of Pa Jin Elmer Co., Ltd and CIS) are closed in the cave; Chemoluminescence agent, for example isoluminol; Sensitiser; Coenzyme; Enzyme substrates; Radio-labeling includes but not limited to
125I,
131I,
35S,
32P,
14C,
3H,
57Co,
99Tc and
75Se, and other radio-labeling as herein described; Particle, for example latex or carbon granule; Metal-sol; Crystallite; Liposome; Cell, etc., but also available dyestuff, catalyzer or the further mark of other detection moiety.Suitable enzyme and coenzyme are disclosed in Litman etc., U.S. Patent number 4,275,149, and Boguslaski etc., U.S. Patent number 4,318,980, each part patent is included this paper by reference in full in.Suitable fluorescent agent and find that luminous agent is disclosed in the Litman that includes in full by reference this paper in etc., U.S. Patent number 4,275,149.Marker also comprises chemical part, but for example can by with specific relevant test section, the vitamin H that detects such as avidin or the Streptavidin combination of mark.Detectable can adopt conventional chemical method known in the art to be connected in antibody of the present invention.
Can marker be produced by many methods can be by the signal of externalist methodology detection, for example by range estimation, electromagnetic radiation, heating and chemical reagent.Described marker also can with another binding members combination in conjunction with antibody of the present invention, or be combined with upholder.
Marker can directly produce signal, therefore, need not other component and produces signal.Many organic molecules, for example fluorescent agent can absorbing ultraviolet and visible light, and the light of absorption is transferred to energy these molecules and they is brought up to excitation level.Then the energy of this absorption dissipates by the light of emission second wave length.The ray of this second wave length also can be transferred to energy the molecule of accepting of mark, and the energy that obtains is by utilizing emitted light, for example FRET (fluorescence resonance energy transfer) (FRET) and dissipating from accepting molecule.Other marker that directly produces signal comprises radio isotope and dyestuff.
Perhaps, marker may need other component to produce signal, signal generation system will comprise the required all components of generation detectable signal so, comprise that substrate, coenzyme, toughener, other enzyme, the material with the reaction of enzyme product, catalyzer, activator, cofactor, inhibitor, scavenging agent, metal ion and binding signal produce the required particular combination material of material.The detailed description of suitable signal generation system is seen Ullman that includes in full this paper with way of reference in etc., U.S. Patent number 5,185,243.
One of binding members, antibody or its function fragment that exists can be the form of immune conjugate, thereby can obtain to detect and/or signal that can be quantitative.Immune conjugate can with the enzyme coupling, for example peroxidase, alkaline phosphatase, α-d-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, N,O-Diacetylmuramidase, malate dehydrogenase (malic acid dehydrogenase) or G 6 PD, or by biological example element, digoxigenin or the coupling of 5-bromouracil deoxyribose equimolecular.Fluorescent marker can be equally and immune conjugate of the present invention or their function fragment coupling; especially comprise fluorescein and derivative thereof, fluorescence dye, rhodamine and derivative thereof, GFP (GFP i.e. " green fluorescent protein "), dansyl base, Umbelliferone, lanthanon sequestrant or cryptate compound, such as europium etc.
Immune conjugate or their function fragment can prepare by method known to those skilled in the art.They can directly or pass through spacer groups or linking group, polyacetals for example, intermediation such as glutaraldehyde, ethylenediamine tetraacetic acid (EDTA) (EDTA), diethylenetriamine pentaacetic acid (DPTA), or at coupling agent, for example described those coupling agents of above therapeutic conjugate are coupled to enzyme or fluorescent marker under existing.The conjugate that contains fluorescein phenotypic marker thing can be by reacting to prepare with lsothiocyanates.Similarly, other immune conjugate can comprise chemiluminescent labels, for example luminol,3-aminophthalic acid cyclic hydrazide and dioxetane, the bioluminescence marker thing, for example luciferase and luciferin, or other radioactively labelled substance, for example iodine 123, I125, iodine 126, iodine 131, iodine 133, bromine 77, Tc 99m, indium 111, indium 113m, gallium 67, gallium 68, S35, P 32, carbon 14, tritium (hydrogen 3), cobalt 57, selenium 75, ruthenium 95, ruthenium 97, ruthenium 103, ruthenium 105, mercury 107, Mercury-203, rhenium 99m, rhenium 101, rhenium 105, scandium 47, tellurium 121m, tellurium 122m, tellurium 125m, thulium 165, thulium 167, thulium 168, fluorine 8, yttrium 199.Those skilled in the art known existing with the therapeutic radiation isotropic substance directly or by sequestrant, for example above-mentioned EDTA, DATA are coupled to the method for antibody, the method can be utilized the used radioelement of diagnosis.Also may pass through chloramine-T method (Hunter and Greenwood 1962) and use Na[I125] mark, or (include in full the U.S. Patent number 4 of this paper in way of reference by the technology of Crockford etc., 424,200) (include in full the U.S. Patent number 4 of this paper in way of reference with the Tc 99m mark or by Hnatowich, 479,930) described continuous through DPTA.Other immune conjugate can comprise toxin moiety, for example is selected from lower group toxin moiety: Pseudomonas exotoxin (PE or its cytotoxin fragment or mutant), diphtheria toxin or its cytotoxin fragment or mutant, botulinum toxin A-F, ricin or its cytotoxin fragment or mutant, abrin or its cytotoxin fragment, sapotoxin albumen or its cytotoxin fragment, the antiviral toxin of Phytolacca acinosa or its cytotoxin fragment and red bryony toxalbumin 1 or its cytotoxin fragment.
Method provided by the invention comprises so that binding members provided herein is combined with IL-17.Should note, this combination can occur in vivo, for example give binding members or the coding binding members nucleic acid after, perhaps can be at ectogensis, for example ELISA, western blotting, immunocytochemistry, immunoprecipitation, affinity chromatography and biological chemistry or test cell line are for example as herein described.The present invention can also utilize binding members of the present invention, for example direct-detection antigen levels in bio-sensor system.
For example, the present invention includes the method in conjunction with situation of detection and/or measurement and IL-17, comprise (i) described binding members is contacted with IL-17 and (ii) detect described binding members and IL-17 in conjunction with situation, wherein adopt the detection of any method described herein or detectable in conjunction with situation.The method and any other as herein described can directly be understood by the personnel that implement the method in conjunction with detection method, for example the visual observations detectable.Perhaps, the method or any other as herein described can produce radioautogram, photo, computer printout output, flow cytometry report, picture, chart, contain any other report visible or the physics manifestation of resultful test tube or container or hole or the method result in conjunction with detection method.
Can measure the binding capacity of binding members and IL-17.Quantitative assay can be relevant with the interested antigen amount of diagnosis in the specimen.Screening IL-17 can be used in conjunction with situation and/or its quantitative assay, for example screens the patient of IL-17 relative disease or illness, and for example its place of this paper is stated.In one embodiment, diagnostic method of the present invention comprises that (i) obtains tissue or the liquid sample of object; (ii) described tissue or liquid sample and one or more binding members of the present invention is contacted; (iii) compare with control sample, detect the steps such as IL-17 of combination, wherein compared with the control, the increase of IL-17 binding capacity can show that IL-17 expresses or activity level is unusual.Tissue to be tested or liquid sample comprise suspects blood, serum, urine, biological biopsy material, tumour or any tissue that contains the IL-17 horizontal abnormality.IL-17 level or active unusual positive object also can be benefited from subsequently disclosed methods for the treatment of of this paper after tested.
Use for reference method disclosed herein, those skilled in the art can be according to their preference and general knowledge Selective determination binding members suitable method of being combined with antigen.
Can be by the reactivity of binding members in any suitable mode working sample.Radioimmunoassay (RIA) is possible a kind of.Radiolabeled antigen mixes with unlabelled antigen (specimen), makes it to be combined with binding members.In connection with antigen and unconjugated antigen physical sepn, measure the radiolabeled antigen amount be combined with binding members.Antigen in the specimen is more, and the radiolabeled antigen of being combined with binding members is fewer.Also the CBA that contains on-radiation antigen be can adopt, the antigen or the analogue that link to each other with reporter molecule utilized.Reporter molecule can be fluorescence dye, phosphorus or the laser dyes with independently spectral absorption or emission characteristic.Suitable fluorescence dye comprises fluorescein, rhodamine, phycoerythrin, texas Red and group of the lanthanides sequestrant or cryptate compound.The suitable dyestuff that adds lustre to comprises diaminobenzidine.
Other reporter molecule comprises macromole colloidal particles or particulate material, for example coloured, magnetic or paramagnetic latex bead and can directly or indirectly produce biology or the chemical active agent of the detectable signal that can pass through visual observations, detected electronically or alternate manner record.For example, these molecules can be enzymes, and its catalyzed reaction is to produce or to change color or cause characteristic electron to change.They can be the molecules that can excite, thereby the transfer transport between the energy level can cause characteristic absorption or emmission spectrum.They can comprise the chemical entities with the biosensor coupling.Can adopt biotin/avidin or vitamin H/Streptavidin and alkaline phosphatase detection system.
Can utilize in the signal acquisition sample that each binding members-reporter molecule conjugate produces (common with test) with binding members in conjunction with situation relevant can quantitatively absolute or relative data.
One aspect of the present invention also provides the test kit of the binding members that comprises any aspect of the present invention or embodiment.In test kit, but the mark binding members, thus can measure its reactivity in sample, for example hereinafter described.Binding members also can with or do not link to each other with solid support.All components of test kit are normally aseptic, are placed in the bottle or other container of sealing.Test kit can be used for diagnositc analysis or utilizes other method of binding members.Test kit can be equipped with in certain method, for example uses the working instructions of these components in the inventive method.Thereby auxiliary substance can be housed in the test kit of the present invention can assist or implement this method.Auxiliary substance comprises the binding members that the second is different, and it can and be coupled to detectable (for example, fluorescent marker, radio isotope or enzyme) in conjunction with the first binding members.Antibody kit also can be equipped with for the pearl of implementing immunoprecipitation.Each component of test kit is contained in the container that is suitable for self usually.Therefore, these test kits comprise the different vessels that is suitable for each binding members usually.In addition, these test kits can be equipped with the working instructions of implementing test and understanding and analyze the method for implementing the test the data obtained.
The present invention also provides the application of antigen levels in the above binding members detection competition experiments, namely utilizes the method for antigen levels in the binding members test sample provided by the invention in competition experiments.This may be to need not the physical sepn combination and situation unconjugated antigen.Reporter molecule may be connected in binding members, thereby the physics of combination or optics change.Reporter molecule can directly or indirectly produce can be quantitative detectable signal.Can be direct or indirect, covalency is for example by peptide bond or non-covalent connection reporter molecule.Connecting via peptide bond may be due to fusion gene recombinant expressed of encoding antibody or reporter molecule.
For example, the present invention includes the method for identifying the IL-17 binding compounds, comprise that (i) is fixed in upholder with IL-17, (ii) described fixing IL-17 is contacted simultaneously or in the substep mode with the test binding compounds of at least a mark, and (iii) new IL-17 binding compounds is identified in the reduction of the combination tag amount of the binding members by the observation mark.
The another kind of method of identifying the IL-17 binding compounds can comprise that (i) is fixed in upholder in connection with the member, (ii) described fixing binding members is contacted simultaneously or in the substep mode with one or more unlabelled test binding members or binding compounds with the IL-17 of mark, and (iii) the combination tag amount of the IL-17 by the observation mark reduce to identify new IL-17 binding compounds.
Can adopt porous or array format, implement these methods in the high-throughput mode.Also can carry out these tests in solution, example as described in Example 2
Test.For example, referring to the U.S.5 that includes in full this paper with way of reference in, 814,468.As mentioned above, in conjunction with detection can be directly understood by the personnel that implement the method, for example by the visual observations detectable, or its amount reduces.Perhaps, binding members of the present invention can produce radioautogram, photo, computer printout output, flow cytometry report, picture, chart, contain any other report visible or the physics manifestation of resultful test tube or container or hole or the method result.
Competition experiments also can be used for epitope mapping.In an example, epitope mapping can be used for identifying the epi-position of IL-17 binding members institute combination, and described binding members is chosen wantonly has neutralization and/or the adjustment feature of optimization.This epi-position can be linear epi-position or conformational epitope.Conformational epitope can comprise at least two different IL-17 fragments, wherein is folded into its three grades or quaternary structure to form the IL-17 inhibitor as IL-17, and for example the position of described fragment is adjoined each other during the conformational epitope of IL-17 binding members identification.In when competition test, can utilize the peptide fragment of antigen, particularly comprise epi-position interested or basically by its peptide that consists of.Can utilize and have epitope sequences and be added with one or more amino acid whose peptides at each end.Binding members of the present invention can be as follows, i.e. the combination of they and antigen is had or the peptide that comprises institute's given sequence suppresses.
The present invention also provides the isolating nucleic acid of code book invention binding members.Nucleic acid can comprise DNA and/or RNA.On the one hand, the invention provides the coding CDR of the invention described above or CDR group or VH structural domain or VL structural domain or antibody antigen-binding site or antibody molecule, for example scFv or IgG are such as the nucleic acid of IgG1.
The present invention also provides plasmid, carrier, transcribe or the construction of expression cassette form, and it comprises at least a above-mentioned polynucleotide.
The present invention also provides the recombinant host cell that comprises above one or more constructions.Encode described CDR or CDR the group or VH structural domain or VL structural domain or antibody antigen-binding site or antibody molecule, for example the nucleic acid of scFv or IgG1 itself consists of an aspect of of the present present invention, this is the same with the method that produces coded product, and the method comprises the expression coding nucleic acid.Cultivating the recombinant host cell that contains this nucleic acid under suitable condition is not difficult to realize expressing.By after express producing, can adopt any suitable technical point from and/or purifying VH or VL structural domain or binding members, then suitably use.
Nucleic acid of the present invention can comprise DNA or RNA, and it can synthesize wholly or in part.Unless statement is arranged in addition, address nucleotide sequence shown in this paper comprise have shown in the dna molecular of sequence, also comprise the RNA molecule of sequence shown in having, wherein U replaces T.
The method that produces antibody VH variable domain is provided in addition on the other hand, and the method comprises the expression coding nucleic acid.The method can be included under the condition that produces described antibody VH variable domain and cultivate host cell.
Producing the VL structural domain is other side of the present invention with the similar approach that comprises the binding members of VH and/or VL structural domain.
The preparation method can comprise the step of separation and/or purified product.The preparation method can comprise product is mixed with and contains at least a other component, for example composition of pharmaceutically acceptable vehicle.
Know the system of in various host cell, cloning and express polynucleotide.The appropriate host cell comprises bacterium, mammalian cell, vegetable cell, filamentous fungus, yeast and rhabdovirus system and transgenic plant and animal.Well known antibody and the antibody fragment of in prokaryotic cell prokaryocyte, expressing.Summary can be referring to Pl ü ckthun 1991.Host bacterium commonly used is intestinal bacteria.
Select as another of preparation binding members, those skilled in the art can also be at the eukaryotic expression of cultivating, for example Chadd and Chamow (2001), Andersen and Krummen (2002), Larrick and Thomas (2001).The mammal cell line that this area can be used for the expressing heterologous polypeptide comprises Chinese hamster ovary (CHO) cell, HeLa cell, baby hamster kidney cell, NS0 mouse melanin tumor cell, YB2/0 rat myeloma cell, human embryonic kidney cell, people's Embryonic Retina cell and many other cells.
The suitable carrier that contains suitable adjusting sequence be can select or make up, promoter sequence, terminator sequence, polyadenylation sequence, enhancer sequence, marker gene and other sequence (if necessary) comprised.Carrier can be plasmid, phagemid for example, or virus, for example phage (if necessary).Other details can referring to, for example Sambrook and Russell (2001).Ausubel 1999 describes many known technologies and the method for operation nucleic acid in detail, for example prepares nucleic acid construct thing, mutagenesis, order-checking, DNA is introduced cell and genetic expression and analysing protein.
Another aspect of the present invention provides the host cell that contains nucleic acid disclosed herein.This host cell can be external and can be in the culture.This host cell can be in vivo.Existence can be expressed as " intracellular antibody " or intrabody in conjunction with being in the molecule with the present invention in the body of host cell.Intracellular antibody can be used for gene therapy.
Provide on the other hand in addition and comprised the method for nucleic acid of the present invention being introduced host cell.Can adopt this introducing of any suitable technology implementation.For eukaryotic cell, suitable technology can comprise calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection and utilize retrovirus or the transduction of other virus that for example vaccinia virus perhaps is baculovirus with regard to insect cell.Nucleic acid is introduced host cell, and particularly eukaryotic cell can utilize virus or pUC pUC.PUC pUC can be maintained at episome and maybe can mix host cell or mix artificial chromosome.Mix can at random or at single or multiple locus place targeted integration a or many parts of copies.For bacterial cell, suitable technology can comprise calcium chloride conversion, electroporation and utilize the bacteriophage transfection.
Expressible nucleic acid after introducing is for example by cultivating host cell under the condition of expressing gene.Can be by the product of method known to those skilled in the art purifying expression.
In one embodiment, nucleic acid of the present invention is integrated into the genome (for example, karyomit(e)) of host cell.According to standard technique, include the sequence that promotes with the genome restructuring in and can promote to integrate.
The present invention also provides to be included in and utilizes above-mentioned construction to express above-mentioned combination and be in or the method for polypeptide in the expression system.
Binding members of the present invention can be designed for diagnosis or treatment human or animal object, for example in people's the method.For example, binding members can be used for diagnosis or treatment IL-17 relative disease or illness, and its example such as this paper state in its place.
Can utilize other example of the illness of binding members treatment of the present invention or diagnosis to comprise:
1. respiratory tract: the occlusive disease of air flue, comprise: asthma, comprise the asthma that (comprising what acetylsalicylic acid and NSAID-brought out) segmental bronchus, supersensitivity, endogenous, exogen, exercise induced, medicine-bring out and dust bring out, intermittently and lasting and all severity, and other inducement of airway hyperreactivity; Chronic obstructive pulmonary disease (COPD); Bronchitis comprises infectivity and eosinocyte bronchitis; Pulmonary emphysema; Bronchiectasis; Cystic fibrosis; Sarcoidosis; Farmer lung and relative disease; Hypersensitivity pneumonitis; Pulmonary fibrosis comprises CFA, idiopathic interstitial lung inflammation, the concurrent antineoplaston of fibrosis and chronic infection, comprises tuberculosis and aspergillosis and other fungi infestation; The complication of lung transplantation; Pulmonary vascular vasculitis and thrombus disease and pulmonary hypertension; Cough-relieving is active, comprises inflammatory chronic cough and the iatrogenic cough relevant with secretion requirement for the treatment of air flue; Acute and chronic rhinitis comprises medicamentous rhinitis and vasomotor rhinitis; Perennial rhinitis and pollinosis comprise nervous rhinitis's (spring fever); Nasal polyposis; Acute viral infection comprises the infection that common cold and respiratory syncytial virus, influenza virus, coronavirus (comprising SARS) and adenovirus cause;
2. bone and joint: osteoarthritis/osteoarthropathy is relevant or comprise the sacroiliitis of osteoarthritis/osteoarthropathy, and the two is primary and insecondary, for example congenital hip dysplasia; The neck lumbar spine is scorching, and waist neck pain; Rheumatoid arthritis and Chauffard-Still disease; The seronegativity spondyloarthropathy comprises ankylosing spondylitis, psoriatic arthritis, reactive arthritis and does not break up SpA (undifferentiatedspondarthropathy); Septic arthritis infects the relevant osteopathia that reaches with other, and for example tuberculosis comprises Pott's disease and Poncet syndrome; The synovitis that acute and chronic crystal brings out comprises uric acid gout, tendon, capsule and synovial membrane inflammation that the calcium phosphate storage disorders is relevant with apatite calcium; Behcet's disease; Primary and Secondary cases xerodermosteosis; Sjogren's syndrome disease and limited scleroderma; Systemic lupus erythematosus, MCTD and undifferentiated connective tissue disease (CTD); Inflammatory myopathy comprises dermatomyositis (dermatomyositits) and polymyositis; Polymyalgia rheumatica (polymalgia rheumatica); Juvenile arthritis comprises idiopathic inflammatory arthritis and relevant syndrome that any joint distributes, and rheumatic fever and general complication thereof; Vasculitis comprises giant cell arteritis, aortic arch syndrome, Qiu-Shi syndrome, polyarteritis nodosa, the vasculitis that micro-polyarteritis is relevant with virus infection, allergy, cryoglobulin and paraprotein; Pain in the back; Familial Mediterranean fever, Mu-Wei syndrome and familial Ireland heat (Familial Hibernian Fever), Kikuchi sick (Kikuchi disease); Drug-induced arthrodynia, tendonitis (tendonititides) and myopathy;
3. because of damage, for example the pain of musculoskeletal disease and reticular tissue are retrofited due to sport injury or the disease: sacroiliitis (for example rheumatoid arthritis, osteoarthritis, gout or crystal joint disease), other joint disease are (for example, cone disk sex change or temporomandibular joint (TMJ) sex change), bone remodeling disease (for example, osteoporosis, Paget's disease or osteonecrosis), polychondritis (polychondritits), scleroderma, MCTD, spondyloarthropathy or periodontopathy (for example, periodontitis);
4. skin: psoriatic, atopic dermatitis, contact dermatitis or other eczema dermatoses and delayed type hypersensitivity; Vegetalitas and solar dermatitis; Seborrheic dermatitis, dermatitis herpetiformis, lichen planus, lichen albus of Von Zumbusch, PG, cutaneous sarcoidosis, discoid lupus erythematosus, pemphigus, pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema venenatum, epidermis eosinophilia, alopecia areata, male pattern baldness, sweet's syndrome, weber-christian syndrome, erythema multiforme; Cellulitis, infectivity and noninfective; Pimelitis; Epidermis lymphoma, nonmelanoma skin cancer and other dysplasia disease damage; Drug-induced disease comprises fixed drug eruption;
5. eyes: marginal blepharitis; Conjunctivitis comprises all the year round and the allergic conjunctivitis in spring; Iritis; Before and posterior uveitis; Choroiditis; Autoimmunization (disease); Affect amphiblestroid sex change or inflammatory diseases; Ophthalmia comprises sympathetic ophthalmia; Sarcoidosis; Infect, comprise that virus, fungus and bacterium infect;
6. gi tract: glossitis, oulitis, periodontitis; Esophagitis comprises anti-stream; Have a liking for eosin gastro-enteritis, mastocytosis, Crohn's disease, colitis, for example ulcerative colitis, Indeterminate colitis, rectitis, micro-colitis, pruritus ani (pruritis ani); Coeliac disease, irritable bowel syndrome, irritability enteropathy, the allergy that non-inflammatory diarrhoea is relevant with food, it may have the effect (for example, migraine, rhinitis or eczema) away from enteron aisle;
7. belly: hepatitis comprises autoimmune hepatitis, alcoholic hepatitis and viral hepatitis; Hepatic fibrosis and liver cirrhosis; Cholecystitis; Pancreatitis, acute and chronic;
8. Genito-urinary: ephritis comprises a matter and glomerulonephritis; Nephrotic syndrome; Urocystitis comprises acute and chronic (matter) urocystitis and hunner's ulcer; Acute and chronic urethritis, prostatitis, epididymitis, ovaritis and salpingitis; Vulvo-vaginitis; The Perun alunite is sick; Erectile dysfunction (masculinity and femininity);
9. allograft refection:, for example after kidney, heart, liver, lung, marrow, skin or the corneal transplantation or the acute and chronic rejection after the blood transfusion; Or chronic graft versus host disease;
10.CNS: alzheimer's disease and other dementia comprise CJD and nvCJD; Amyloidosis; Multiple sclerosis and other demyelination syndrome; Cerebral atherosclerosis and vasculitis; Temporal arteritis; Myasthenia gravis; Acute and chronic pain (acute, intermittence or persistence, nervus centralis or peripheral nerve source no matter), comprise pain, neuropathic pain syndrome that Encelialgia, headache, migraine, trigeminal neuralgia, atypia prosopodynia, joint and bone pain, cancer and tumor invasion produce, comprise the neuropathy relevant with HIV-after diabetes, the bleb; Neurosarcoidosis; The central nervous system of pernicious, infectivity or the self-immunprocess peripheral nervous system complication of unifying;
11. other autoimmunization and anaphylactic disease comprise Hashimoto thyroiditis, Robert Graves (family name) disease, A Disen (family name) disease, diabetes, primary thrombocytopenic purpura, eosinophilic fasciitis, height-IgE syndrome (hyper-IgE syndrome), antiphospholipid syndrome;
12. have the Other diseases of inflammatory or immunology component: comprise acquired immune deficiency syndrome (AIDS) (AIDS), leprosy, Sezary syndrome and paraneoplastic syndrome (paraneoplastic syndrome);
13. cardiovascular: atherosclerosis affects coronary artery and peripheral circulation; Pericarditis; Myocarditis, inflammatory and autoimmune cardiomyopathy comprise myocardial sarcoisosis; The ischemic reperfusion injury; Endocarditis, cardiovalvulitis, and aortitis comprise infectivity (for example syphilis); Vasculitis; The disease of proximal venal and peripheral vein comprises phlebitis and thrombosis, comprises that dvt forms and the varix complication; With
14. oncology: treat common cancer, comprise prostate gland, mammary gland, lung, ovary, pancreas, intestines and colon, stomach, skin and cerebral tumor and affect the malignant tumour of marrow (comprising leukemia) and lymphadenosis system, for example Huo Qijin and non-Hodgkin lymphoma; Comprise the other syndrome of prevention and treatment metastatic disease and tumor recurrence and cancer.
Therefore, binding members of the present invention can be used as the therapeutical agent that treatment relates to the illness of unusual IL-17 expression and/or activity.A kind of embodiment is a kind of methods for the treatment of, comprises the patient who the binding members of the present invention of significant quantity is needed it, wherein the unconventionality expression of IL-17 and/or activity decreased.Another kind of embodiment is a kind of methods for the treatment of, comprise (i) assay certificate IL-17 level or active unusual patient, for example adopt above-mentioned diagnostic method, and (ii) give this patient with the binding members of the present invention of significant quantity, wherein the unconventionality expression of IL-17 and/or activity decreased.According to the present invention, significant quantity is to reduce the unconventionality expression of IL-17 and/or active consumption, thus can reduce or alleviate at least a symptom of the disease for the treatment of or illness, but need not to cure this disease or illness.Therefore, an embodiment of the invention are methods of the severity for the treatment of or at least a symptom that alleviates any disease described herein, comprise one or more binding members of the present invention of significant quantity are united the patient that these needs are arranged separately or with combined therapy scheme and another kind of suitable drug known in the art or as herein described, thereby can reduce the severity of at least a symptom of described disease.Another embodiment of the invention is the method for at least a effect of antagonism IL-17, comprise one or more binding members of the present invention that contact or give significant quantity, thereby the described at least a effect of energy antagonism IL-17, for example IL-17 is combined or downstream effect with IL-17RA, for example release of cytokines.
Therefore, other side of the present invention provides methods for the treatment of, the binding members that gives to provide, the pharmaceutical composition that comprises this binding members are provided, and the application of this binding members in the medicine that preparation gives, the method that for example prepares medicine or pharmaceutical composition comprises with pharmaceutically acceptable vehicle preparation binding members.Pharmaceutically acceptable vehicle can be the combination of compound or compound, it can be included pharmaceutical composition in but can not cause secondary response and energy, for example promote giving of active compound, increase its volume lifetime and/or its and render a service, increase its in solution solubleness or prolong its storage time.These pharmaceutically acceptable carriers are known, and those skilled in the art can improve character and the administering mode of these carriers to adapt to selected active compound.
Binding members of the present invention gives with the form of pharmaceutical composition usually, and described composition can comprise except at least a composition that is combined into an ancient official title.Therefore, for being used for the present invention, except activeconstituents, pharmaceutical composition of the present invention can comprise pharmaceutically acceptable vehicle, carrier, damping fluid, stablizer or other material well known to those skilled in the art.These materials should be nontoxic, do not answer the effectiveness of interferon activity composition.The precise nature of carrier or other material depends on route of administration, its can be oral, suck or through injection, for example intravenous administration.
The present invention has also considered the oral pharmaceutical composition that gives, such as nanometer body (nanobody) etc.This oral preparations can be tablet, capsule, powder, liquid or semi-solid form.Tablet can comprise solid carrier, for example gelatin or adjuvant.Composition of liquid medicine comprises liquid vehicle usually, for example water, oil, animal or plant oil, mineral oil or synthetic oil.Can comprise physiological salt solution, glucose or other sugar soln or glycerine, for example ethylene glycol, propylene glycol or polyoxyethylene glycol.
For intravenous injection, or in the patient part injection, activeconstituents can be the acceptable aqueous solution form of parenteral, and it does not contain pyrogen, and pH, isotonicity and stability are suitable.The relevant technical staff in the field can utilize, and oozes carrier such as waiting, and for example sodium chloride injection, ringer's injection, lactated Ringer's injection liquid prepare suitable solution.If necessary, can utilize sanitas, stablizer, damping fluid, antioxidant and/or other additive, comprise damping fluid, for example phosphoric acid, citric acid, Histidine and other organic acid; Antioxidant, for example xitix and methionine(Met); Sanitas (stearyl dimethyl benzyl ammonium chloride for example; Chloor-hexaviet; Benzalkonium chloride, Solamin; Phenol, butanols or benzylalcohol; P-hydroxybenzoic acid alkane ester, for example methyl p-hydroxybenzoate or propyl ester; Catechol; Resorcinol; Hexalin; 3 '-amylalcohol; And m-cresol); Low molecular weight polypeptide; Protein, for example serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, for example polyvinylpyrrolidone; Amino acid, for example glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrate comprise glucose, seminose or dextrin; Sequestrant, for example EDTA; Sugar, for example sucrose, mannitol, trehalose or sorbyl alcohol; Salify counter ion, for example sodium; Metal complex (for example, Zn-protein complex); And/or nonionogenic tenside, for example TWEEN
TM, PLURONICS
TMOr polyoxyethylene glycol (PEG).
According to physicochemical property and the route of delivery of molecule, binding members of the present invention can be mixed with liquid, semisolid or solid form.Preparation can comprise the combination of vehicle or vehicle, for example sugar, amino acid and tensio-active agent.The antibody concentration that liquid preparation comprises and pH wide range.Can pass through, for example lyophilize, spraying drying or supercritical liq technology drying prepare solid preparation.The preparation of anti--IL-17 depends on required route of delivery: for example, the preparation of pulmonary delivery can be made of particle, and its physical property is guaranteed can penetrate the lung deep after suction; Topical formulations can comprise the viscosity modifying agent of energy prolong drug in the site of action residence time.In some embodiments, the available carrier that can prevent that binding members from discharging fast prepares binding members, and for example controlled release preparation comprises implant, transdermal patch and microencapsulation delivery system.Can utilize biodegradable, biocompatible polymkeric substance, for example ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, poe and poly(lactic acid).Many methods of known these preparations of preparation of those skilled in the art.Referring to, Robinson for example, 1978.
Can be oral (for example, the nanometer body), by injection (for example, subcutaneous, intraarticular, intravenously, intraperitoneal, intra-arterial or intramuscular), by suck, by approach (being instilled into bladder) in the vesica or by local (for example, in intraocular, the nose, rectum, enter wound or give on the skin) utilize binding members of the present invention anti--IL-17 treats.Can be by the pulse infusion, the binding members that particularly gradually falls with dosage is treated.Can render a service or the requirement that reduces as far as possible side effect decides route of administration by the physicochemical characteristics for the treatment of, concrete Consideration or optimize by disease.A kind of concrete route of administration is intravenously.The another kind of approach that gives pharmaceutical composition of the present invention is subcutaneous.Estimate that anti--IL-17 treatment can not be confined to use in the clinic.Therefore, preferably utilize the subcutaneous injection of needle-less device.
According to disease to be treated, composition can be separately or with other therapeutic combination, simultaneously or successively or the conduct compound artifact that contains another kind of therapeutical agent or various medicaments give.
The binding members of IL-17 can be used as the part with the combined therapy of other medicines component coupling.Can adopt combined therapy that obvious synergy is provided, particularly the combination of anti--IL-17 binding members and one or more other medicines.With regard to treating one or more diseases as herein described, the binding members of IL-17 can give simultaneously or successively or as containing the compound artifact of another kind of therapeutical agent or various medicaments.
The form that can make up or add one or more following medicaments provides binding members of the present invention:
The antagonist of-cytokine function (for example, acting on the medicament of cytokine signaling pathway, for example the conditioning agent of SOCS system), for example α-, β-and/or gamma-interferon; I type rhIGF-1 (IGF-1), the conditioning agent of its acceptor and Binding proteins; Interleukin (IL), for example one or more among the IL-1-33, and/or interleukin antagonist or inhibitor, for example Kineret; The inhibitor of the inhibitor of interleukin family member's acceptor or the specific subunit of these acceptors; Tumor necrosis factor alpha (TNF-α) inhibitor, for example anti-TNF monoclonal antibody (for example, infliximab; Adalimumab and/or CDP-870) and/or TNF receptor antagonist, for example immunoglobulin molecules (for example etanercept) and/or lower molecular weight medicament, for example pentoxifylline (pentoxyfylline);
-B cell modulator, target B-lymphocyte (CD20 (Rituximab) or MRA-aIL16R) or the monoclonal antibody of T-lymphocyte (for example, CTLA4-Ig, HuMax Il-15 or Orencia (Abatacept)) for example for example;
The conditioning agent of-inhibition osteoclast activity, for example antibody of RANKL;
The conditioning agent of-chemokine or chemokine receptor function, for example antagonist of following chemokine: CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (with regard to C-C family); CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 and CXCR6 (with regard to C-X-C family) and C-X
3The CX of-C family
3CR1;
-matrix metalloproteinase (MMP), i.e. following one or more inhibitor: stromelysins, collagenase and gelatinase and aggrecan enzyme; Particularly collagenase-1 (MMP1), collagenase-2 (MMP8), collagenase-3 (MMP13), stromelysins-1 (MMP3), stromelysins-2 (MMP10) and/or stromelysins-3 (MMP11) and/or MMP9 and/or MMP12 are such as medicaments such as doxycyclines;
-inhibitors of leukotriene biosynthesis, 5-lipoxygenase (5-LO) inhibitor or 5-lipoxygenase activated protein (FLAP) antagonist, for example: zileuton; ABT-761; Fenleuton; Tepoxalin; Abbott-79175; Abbott-85761; N-(5-replaces)-thiophene-2-alkyl sulfonamide; The 2,6 di t butyl phenol hydrazone; Methoxyl group tetrahydropyrans, for example zeneca ZD-2138; Compound S B-210661; The 2-cyano group naphthalene compound of pyridyl-replacement, L-739 for example, 010; 2-cyano quinolines compound, L-746 for example, 530; Indoles and/or quinoline compound, for example MK-591, MK-886 and/or BAY x 1005;
The receptor antagonist of-leukotriene (LT) B4, LTC4, LTD4 and LTE4 is selected from: thiodiphenylamine-3-1s, L-651 for example, 392; Amidino compounds, for example CGS-25019c; Amino benzoxazoles class (benzoxalamines), for example Ontazolast; Benzamidine class (benzenecarboximidamides), for example BIIL 284/260; With compounds such as Zafirlukast, Ro 23-3544, Singulair, pranlukast, verlukast (MK-679), RG-12525, Ro-245913, iralukast (CGP 45715A) and BAY x 7195;
-phosphodiesterase (PDE) inhibitor, methyl xanthine (methylxanthanine) for example is such as theophylline and/or aminophylline; And/or selectivity PDE isozyme inhibitor, for example inhibitor of PDE4 inhibitor and/or isotype PDE4D, and/or PDE5 inhibitor;
-1 type histamine receptor antagonists, for example alerlisin, Loratadine, Desloratadine, fexofenadine, acrivastine, terfenadine, astemizole, azelastine, Levocabastine, chlorphenamine, promethazine, marezine and/or mizolastine (usually oral, base portion or parenteral application);
-proton pump inhibitor (for example omeprazole) or stomach protectiveness histamine 2 receptor antagonist;
-histamine 4 receptor antagonists;
-α-1/ α-2 adrenoceptor agonists, vasoconstrictor, sympathomimetic, for example hexahydrodesoxyephedrine, phenylephrine, Phenylpropanolamine, racephedrine, pseudoephedrine, naphcon, Oxymetazoline Hydrochloride, tetrahydrozoline hydrochloride, xylometazoline hydrochloride, tramazoline hydrochloride and ethylnorephinephrine hydrochloride;
-anticholinergic drug, for example M-ChR (M1, M2 and M3) antagonist, for example coromegine, Scopolamine, Tarodyn (glycopyrrrolate), ipratropium bromide, tiotropium bromide, oxitropium bromide, pirenzepine and telenzepine;
-receptor,β agonist (comprising beta receptor hypotype 1-4), for example Racemic isoproterenol, salbutamol, formoterol, Salmeterol, terbutaline, Metaprel, Bitolterol Mesylate and/or pirbuterol, for example their chirality enantiomorph;
-chromone, for example Sodium Cromoglicate and/or sodium nedocromil;
-glucocorticosteroid, for example flunisolide, Triamcinolone Acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate, ciclesonide and/or furoic acid momisone;
The medicament of-adjusting nuclear hormone receptor, for example a PPAR;
Antagonist or the antibody of-immunoglobulin (Ig) (Ig) or Ig goods or adjusting Ig function, for example anti--IgE (for example, Ao Mali pearl monoclonal antibody (omalizumab));
The antiphlogiston of-other general or topical application, for example Thalidomide or derivatives thereof, retinoid, anthratriol and/or calcipotriol;
The combination of-aminosalicylate and sulfapyridine, for example sulfasalazine, mesalazine, Balsalazide and olsalazine; Immunomodulator, for example purinethol (thiopurines) and reflunomide, for example budesonide;
-antiseptic-germicide, for example penicillin derivative, tsiklomitsin, Macrolide, beta-lactam, fluoroquinolone, metronidazole and/or imbedibility aminoglycoside; And/or antiviral agent, for example aciclovir, Famciclovir, valaciclovir, ganciclovir, cidofovir; Adamantanamine, Rimantadine; Ribavirin; Zanamivir and/or oseltamivir; Proteinase inhibitor, for example indinavir, nelfinavir, ritonavir and/or Saquinavir; Nucleoside reverse transcriptase inhibitor, for example Didanosine, lamivudine, stavudine, zalcitabine, zidovudine; Non-nucleoside reverse transcriptase inhibitor, for example nevirapine, efavirenz;
-cardiovascular agent, for example calcium channel blocker, receptor,β blocker, angiotensin-converting enzyme (ACE) inhibitor, angiotensin-2 receptor antagonist; Lipid lowering agent, for example special class of Statins and/or shellfish; The conditioning agent of blood cell shape, for example pentoxifylline; Thrombus and/or antithrombotics, for example hemocyte aggregation inhibitor;
-CNS medicine, thymoleptic (for example Sertraline) for example, anti--Parkinson's medicine (L-deprenyl for example, levodopa, ropinirole, pramipexole, the MAOB inhibitor, such as Sai Lejin (selegine) and rasagiline, the comP inhibitor, such as tolcapone, the A-2 inhibitor, dopamine reuptake inhibitor, nmda antagonist, nicotinic agonist, dopamine agonist and/or neuronal nitric oxide synthase inhibitor) and anti-Alzheimer disease medicine, for example E2020, rivastigamine, tacrine, cox 2 inhibitor, propentofylline or Metrifonate;
The medicament of the acute and chronic pain of-treatment, the for example analgesic agent of maincenter or peripheral action, for example opium analogue or derivative, kappa rice piperazine, Phenytoin Sodium Salt, Sodium Valproate, amitriptyline (amitryptiline) or other thymoleptic, paracetamol or nonsteroid anti-inflammatory drugs;
(comprising suction) local anesthetic, for example lignocaine or its analogue of-parenteral or part-application;
-anti--osteoporosis agent, hormonal medicaments for example is such as raloxifene or diphosphonate, such as alendronate;
-(i) trypsin inhibitor; (ii) hemocyte activation factor (PAF) antagonist; (iii) interleukin converting Enzyme (ICE) inhibitor; (iv) IMPDH inhibitor; (v) adhesion molecule inhibitor comprises the VLA-4 antagonist; (vi) kethepsin; (vii) kinase inhibitor, for example tyrosine kinase inhibitor (for example, the example of Btk, Itk, Jak3 MAP inhibitor can comprise Gefitinib (Gefitinib), imatinib mesylate), serine/threonine kinase (for example, the inhibitor of map kinase, such as p38, JNK, protein kinase A, B and C and IKK) or participate in the kinases (for example, relying on the kinases of cyclin) of Cycle Regulation; (viii) glucose-6 monophosphate dehydrogenase inhibitor; (ix) kassinin kinin-B
1-and/or B
2-receptor antagonist; (x) anti--gout medicament, for example colchicine; (xi) xanthine oxidase inhibitor, for example Zyloric; (xii) uricosuric agent, for example, probenecid, sulfinpyrazone and/or benzbromarone; (xiii) growth hormone cinogenic agent; (xiv) transforming growth factor (TGF β); (xv) platelet-derived growth factor (PDGF); (xvi) fibroblast growth factor, for example Prostatropin (bFGF); (xvii) rHuGM-CSF (GM-CSF); (xviii) capsicine emulsifiable paste; (xix) tachykinin NK-1
1And/or NK
3Receptor antagonist, for example NKP-608C, SB-233412 (Talnetant) and/or D-4418; (xx) elastase inhibitor, for example UT-77 and/or ZD-0892; (xxi) TNF-α converting enzyme inhibitor (TACE); (xxii) inducible nitric oxide synthase (iNOS) inhibitor or (xxiii) the chemoattractant acceptor-homolgous molecule of expression on the TH2 cell, (for example, CRTH2 antagonist); (xxiv) inhibitor of P38; (xxv) medicament of the function of adjusting Toll-sample acceptor (TLR) and (xxvi) medicament of adjusting purinergic receptor activity, for example P2X7; (xxvii) inhibitor of transcription factor activation, for example NFkB, API and/or STATS.
Inhibitor can be that specific inhibitor maybe can be mixed-type inhibitor, for example the inhibitor of the above-mentioned different kinds of molecules of target (for example acceptor) or molecule type.
Binding members can also give or form and chemotherapeutics or the another kind of tyrosine kinase inhibitor coupling of immune conjugate jointly.The non-fragment of described antibody also can be used in the bi-specific antibody that obtains by recombination mechanism or biological chemistry coupling, thereby can be in conjunction with the specificity of above-mentioned antibody and the specificity of other antibody, described other antibody capable identification relates to relevant other the active molecule of IL-17.
For the treatment inflammatory diseases, binding members of the present invention can make up with one or more medicaments, for example
-nonsteroid anti-inflammatory drugs (hereinafter referred to as NSAID), comprise non-selectivity cyclo-oxygenase (COX)-1/COX-2 inhibitor, no matter part or general are used (for example piroxicam, diclofenac, propionic acid, such as naprosine, flurbiprofen, fenoprofen, Ketoprofen and Ibuprofen BP/EP, fragrant that ester, such as mefenamic acid, indomethacin, sulindac, Azapropazone, pyrazolone, for example BUTE, salicylate, for example acetylsalicylic acid); Selective COX-2-2 inhibitor (for example, meloxicam, celecoxib, rofecoxib, valdecoxib, Lu Makao former times (lumarocoxib), parecoxib and L-791456); The cyclo-oxygenase (CINODs) that suppresses nitric oxide donors; Glucocorticosteroid (no matter by local, oral, intramuscular, intravenously or intraarticular approach); Methotrexate, leflunomide; Oxychloroquine, d-Trolovol, auranofin or other parenteral or New Oral Gold goods; Analgesic agent; Diacerein; Intraarticular treatment, for example derivatives of hyaluronic acids; And nutritional additive, for example glycosamine.
Binding members of the present invention also can use with the existing therapeutic combination for the treatment of cancer.The suitable medicament of use capable of being combined comprises:
(i) be used for the antiproliferative/antitumor drug of Medical oncology and their combination, for example alkylating agent (for example, cis-platinum, Carboplatin, endoxan, mustargen, melphalan, Chlorambucil, busulfan and nitrosourea); (for example, antifol is such as 5-FU, such as 5 FU 5 fluorouracil and Tegafur, Raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, gemcitabine and taxol for metabolic antagonist; Antitumor antibiotics (for example, anthracycline antibiotics is such as Zorubicin, bleomycin, Dx, daunomycin, epirubicin, idarubicin, Mitomycin-C, gengshengmeisu and Plicamycin); Anti-(having silk to divide) splits agent (for example, vincaleucoblastine is such as vincristine(VCR), vinealeucoblastine(VLB), vindesine and vinorelbine and Taxan, such as taxol and taxotere); And topoisomerase enzyme inhibitor (for example, Zuyeyidal is such as Etoposide and teniposide, amsacrine, Hycamtin and camptothecine);
(ii) cytostatic, for example estrogen antagonist (for example, tamoxifen, toremifene, raloxifene, droloxifene and indoles former times fragrant (iodoxyfene)), (for example adjust under the estrogen receptor, fulvestrant), androgen antagonist (for example, bicalutamide, flutamide, Nilutamide and cyproterone acetate), lhrh antagonist or LHRH agonist are (for example, goserelin, Leuprolide and buserelin), progestogen (for example, Magace), aromatase inhibitor (for example, Anastrozole, letrozole, vorozole (vorazole) and Exemestane) and the inhibitor of 5α-reductase, for example finasteride;
(iii) medicament (for example, inhibitors of metalloproteinase is such as the inhibitor of Marimastat and UPA function of receptors) of anticancer invasion and attack;
(iv) inhibitor of somatomedin function, for example, this inhibitor comprises growth factor antibodies, growth factor receptor antibody (for example, anti--erbb2 antibody, Herceptin and anti--erbb1 antibody Cetuximab [C225]), farnesyl transferase inhibitor, tyrosine kinase inhibitor and serine/threonine kinase inhibitor, for example, the epidermal growth factor family inhibitor (for example, EGFR family tyrosine kinase inhibitor, such as N-(3-chloro-4-fluorophenyl)-7-methoxyl group-6-(morpholinyl propoxy-) quinazoline-4-amine (Gefitinib, AZD1839), N-(3-ethynyl phenyl)-6,7-two (2-methoxy ethoxy) quinazoline-4-amine (erlotinib (erlotinib), OSI-774) and 6-propenyl amido-N-(3-chloro-4-fluorophenyl)-7-(morpholinyl propoxy-) quinazoline-4-amine (CI 1033)), for example, the inhibitor of platelet-derived growth factor family and for example, the inhibitor of pHGF family;
(v) angiogenesis inhibitor medicament, those medicaments that for example suppress the effect of vascular endothelial growth factor, (for example, anti-vascular endothelial growth factor antibody Avastin, International Patent Application WO 97/22596, WO97/30035, WO 97/32856 and WO 98/13354 disclosed compound, each part full patent texts is included this paper in) and the compound (for example, inhibitor and the angiostatin of linomide, beta 2 integrin alpha v β 3 functions) that works with other mechanism;
(vi) vascular damaging agents, disclosed compound among combretastatin A4 and International Patent Application WO 99/02166, WO00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and the WO 02/08213 for example, each part full patent texts is included this paper in;
(vii) antisense therapy, for example, for those of above-mentioned target, such as ISIS 2503, anti--the ras antisense;
(viii) gene therapy method, for example comprise, substitute aberrant gene, method, GDEPT (the enzyme prodrug treatment that gene instructs) method such as unusual p53 or unusual BRCA1 or BRCA2, for example utilize those methods of Isocytosine deaminase, thymidine kinase or bacterium nitroreductase and increase the patient to the method for chemotherapy or radiotherapy tolerance, for example multi-medicine resistance gene therapy; With
(ix) immunotherapy method, for example comprise, exsomatize with the interior method of body to increase the immunogenicity of patient tumors cell, as use cytokine, such as interleukin-22, IL-4 or rHuGM-CSF transfection, reduce the method for T cellular energy, utilize the immunocyte of transfection, the method of the dendritic cell of cytokine transfection for example, utilize cytokine transfection tumor cell line method and utilize the method for antiidiotypic antibody.
Binding members of the present invention and one or more above-mentioned other medical science components can be used for preparing medicine.Medicine can give individuality alone or in combination, therefore can comprise binding members and other component, for example compound artifact or independent goods.Independent goods can be used for promoting separately and successively or simultaneously administration, and can pass through different approaches, for example orally give all components with parenteral admin.
According to the present invention, the composition that provides can give Mammals.Administration can be " treatment significant quantity ", and this is enough to show benefit for the patient.This benefit can be alleviated at least a symptom at least.The actual amount that gives and injection speed and time-histories depend on character and the severity of the disease for the treatment of, the concrete Mammals for the treatment of, each patient's clinical disease, the inducement of disease, the site of delivery of composition, the type of binding members, medication, administration time table and the known other factors of medical science opening personnel.Common opening personnel and other doctor are responsible for treatment prescription, and such as the decision of dosage etc., it depends on severity and/or the progress of the disease symptoms for the treatment of.Well known suitable antibody dosage (Ledermann 1991 and Bagshawe1991).Can utilize the concrete dosage that is suitable for the described herein of the drug type that gives or Physician ' s DeskReference (doctor's desk reference) (2003).Can measure by in animal model, comparing its external activity and activity in vivo treatment significant quantity or the appropriate dose of binding members of the present invention.The method of the known effective dose of in mouse and other test animal, extrapolating.Exact dosage desired depends on many factors, comprise that antibody is for diagnosis, prevention or treatment, the size of therapeutic area and position, the character of the precise nature of antibody (for example, whole antibody, fragment or double antibody) and any detectable or other molecule of linking to each other with antibody.For the general administration, classical antibody dosage is 100 μ g-1g, is 1 μ g-1mg for topical application.Can give first higher loading dosage, then give one or many than low dosage.Antibody generally is whole antibody, for example the IgG1 isotype.This is the dosage of adult patients single therapy, can appropriately adjust for children and baby, also can regulate pro rata according to molecular weight for other antibody formation.Treatment can follow the doctor's advice with once a day, every kind twice, every kind once or mensal interval repeat.Treatment can be that all subcutaneous the giving of every 2-4 give with every 4-8 state intravenously.In some embodiments of the present invention, treatment is periodic, is about for two weeks or longer period between the administration, and for example about 3 weeks or longer, about 4 is all or longer, or approximately per month once.In other embodiment of the present invention, treatment can give before surgical operation and/or afterwards, can directly give or be applied to the region of anatomy of surgical operation therapy.
Embodiment
Embodiment 1: the separation of antibody leading (Antibody lead)
1.1 select
Utilization is cloned into selects according to the large library of the scFv of the phagemid vector of filobactivirus M13 (scFv) people's antibody that (Vaughan etc. 1996; Hutchings etc. 2001).Utilizing basically, aforesaid recombinant human IL-17A carries out how wheel is selected to separate anti--IL-17A specificity scFv antibody (Vaughan etc. 1996) from phage display library.In brief, 4 ℃ of human il-17s with PBS (Dulbecco PBS, pH7.4) preparation are adsorbed on each hole of microtiter plate, spend the night.Wash each hole with PBS, then use PBS-Marvel (3%w/v) sealing 1 hour.The purifying phage of PBS-Marvel (3%w/v) preparation is added each hole, make it to be combined 1 hour with coated antigen.With PBS-tween (0.1%v/v) and the unconjugated phage of PBS continuous washing.The phage particle of elution of bound infects bacterium and rescue and selects (Vaughan etc. 1996) to carry out next round.
Making second of representative number take turns respectively being cloned in the 96-orifice plate of selection grows.ScFv expresses in the bacterium pericentral siphon, adopts human il-17 acceptor A active in conjunction with their inhibition of experiment sieving.Interaction shows that obvious inhibiting scFv (as the pericentral siphon crude extract) carries out dna sequencing (Vaughan1996, Osbourn 1996) to IL-17A:IL-17RA.Unique scFv expresses in bacterium again, affinitive layer purification (Bannister etc. 2006), by
Receptor-ligand is measured IC in conjunction with test with for the serial dilution that detects purifying scFv in the HT1080 IL-6 release test of people and stump-tailed macaque IL-17
50Value.
1.2
The selectivity of antibody and species cross reactivity in the epi-position competition experiments
Adopt
The epi-position competition experiments, determine species cross reactivity and the selectivity of IL-17 family member's antibody by detection and the inhibition degree of the HIS label IL-17A (HIS FLAG IL-17A) (inside, HEK EBNA derives) of each anti--IL-17 antibodies of fixing.
Test unlabelled Purification of Human IL-17A (inside, intestinal bacteria derive), IL-17B (Pai Puluo technology company (Peprotech)), the IL-17C ((R of RD system house in each test; D Systems)), the titre of IL-17D (Pai Puluo technology company), IL-17E (RD system house) and IL-17F (Pai Puluo technology company) is to determine each IL-17 family member's effectiveness, such as IC in the test
50Survey.
Test comprises that people, stump-tailed macaque (inside, intestinal bacteria derive) and Muridae IL-17A (RD system house) are at the titre of the interior various IL-17 kinds species cross reactivity with definite these antibody in each test.
IgG with purifying in phosphate-buffered saline (PBS) is adsorbed onto on the 96-hole Maxisorp microtiter plate (Nunc), for this concrete IgG, estimate that when biotinylated human il-17 A being shown greatly it Kd adds fashionable its concentration and can obtain obvious signal.With the excessive IgG of PBS-tween (0.1%v/v) flush away, sealed each hole 1 hour with PBS-Marvel (3%w/v).The serial dilution for preparing rival (Muridae or stump-tailed macaque IL-17A or IL-17 family member) in PBS, its concentration start from about 200 times of Kd value of biotinylation human il-17 A and IgG interaction separately.Not biotinylated human il-17 A is as positive control.Adding concentration in this serial dilution is Kd2 equal-volume biotinylation recombinant human IL-17A (obtain competition antigen: the initial ratio of biotinylation human il-17 A is about 100: 1 serial dilution) doubly approximately.Then these mixtures are transferred on the IgG of sealing balance 1 hour.Remove unconjugated antigen with PBS-tween (0.1%v/v) washing, and with Streptavidin-europium 3+ conjugate (
Detect Pa Jin Elmer Co., Ltd (PerkinElmer)) the remaining biotinylation human il-17 A of detection.Utilize the dull and stereotyped reader of EnVision (Pa Jin Elmer Co., Ltd) to detect the time meta-resolution fluorescence of 620nm.With fluorescence data be converted to the % specific binding (100% from containing biotinylation human il-17 A but the control wells that does not contain the rival measure, 0% measures from the hole of containing biotinylation human il-17 A and surpassing 100 times not biotinylation human il-17 A).The data that the Prism curve fitting software analysis of utilization figure pad company obtains are to measure IC
50Value.Because each IgG is different to the avidity of IL-17A, should adopt different test conditionss, the result is expressed as IC between human il-17 A and the competition antigen
50The difference multiple of value.Thereby IgG that can be more different.
Table 1: human il-17 A tests in conjunction with IgG
The effectiveness of middle IL-17 family member and different I L-17 kind
ND shows undetermined, the NI unrestraint.Any clone for test does not observe IL-17 B-F human il-17 A is combined with restraining effect.
1.3 suppress IL-17A and IL-17 receptors bind
With the receptor-ligand combination
(homogenizing time-resolution fluorescence, the biological international corporation (CISBio international) of CIS) test form screening selection result is to the human il-17 A (inside, HEK EBNA derives) of biotinylation human il-17 A (200-17 of Pai Puluo technology company) or the HIS label restraining effect in conjunction with IL-17RA Fc fusion rotein (177-IR of RD system house).In all potency tests, comprise reference anti--IL-17mAb (biogenic company (Biosource)) is as positive control.Detailed test method is seen test materials and method chapters and sections.
Table 2 has shown the example of testing the leading scFv effectiveness that obtains from the human il-17 A of HIS label in conjunction with IL-17RA Fc fusion rotein.
The example that the leading scFv that the IL-17A of table 2:HIS label obtains in conjunction with the test of IL-17RA Fc fusion rotein renders a service
1.4 epi-position combination
The leading scFv of test is to HIS label human il-17 A (inside in the epi-position competition experiments, HEKEBNA derives) in conjunction with the Anti-Human IL-17 molecule restraining effect of (comprising parental generation antibody 1, the mAb of mAb317 (RD system house) or biogenic company).
Utilize the epi-position competition experiments of the mAb of mAb317 or biogenic company comprise with 3nM or 6nM anti--IL-17 antibody and 20nM or 10nM XL665 mark anti--Muridae Fc (the CIS biology 61PAMXLB of international corporation) preincubation in the 50mMHEPES that contains respectively 0.4M Potassium monofluoride and 0.1%BSA (test damping fluid).Antibody and XL665 detect (system) at room temperature lucifuge preincubation 1 hour.For the test that utilizes antibody 1, with the direct traget antibody 1 of cryptate compound (the biological 62EUSPEA of international corporation of CIS), and re-use with 500 times of test damping fluid dilutions first.
Simultaneously, for the test of the mAb that utilizes mAb317 or biogenic company, under the room temperature with the biotinylated IL-17A of 10nM (20nM monomer) and 3.2nM Streptavidin cryptate compound lucifuge incubation 1 hour in the test damping fluid.For the test that utilizes antibody 1, under the room temperature with 2nM HIS label IL-17A (4nM monomer) and 2nM XL665 mark anti--tag antibody (the biological 61FG2XLB of international corporation of CIS) preincubation 1 hour in the test damping fluid.
After the reagent preincubation, the scFv sample of 10 μ l purifying is added the low volume test panel (Rodrigo Costa company (Costar)) in 384 holes.For the test that utilizes mAb317 or biogenic mAb, the IL-17 antibody and the XL665 thereof that add subsequently 5 μ l preincubation detect (system), then add 5 μ l people biotinylation IL-17A and cryptate compound and detect mixture.
For antibody 1 test, the scFv sample of 10 μ l purifying is added the low volume test panel in 384 holes, then add the antibody 1 of 5 μ l cryptate compound marks, add again 5 μ l HIS label IL-17A and XL665 and detect mixture.
Then at room temperature with test panel with 1000rpm centrifugal 1 minute, incubation is 4 hours under the room temperature, uses the dull and stereotyped reader of EnVision (Pa Jin Elmer Co., Ltd) to read the time meta-resolution fluorescence of 620nm and 665nm emission wavelength again.
Come analytical data by the % Δ F value (the biological international corporation of CIS) of calculating each sample, then be used for measuring the % specific binding.
The results are summarized in table 3.
Table 3: the example of leading scFv characterization result in IL-17A epi-position competition experiments
It is good that +++observe suppressed
+ to observe inhibition not good
-do not observe inhibition
In 3 epi-position competition experiments, observe inhibition in various degree between the leading group of objects of scFv, thereby show that among these clones some identify the different or overlapping epi-position of other scFv and the mAb that sets up these tests in this group objects slightly.
1.5 being changed (reformatting), scFv becomes IgG1
By with V
HAnd V
LThe structural domain Asia is cloned into respectively the carrier of the heavy chain of antibody of the expressed and light chain and will clones from scFv and converts the IgG form to.With V
HStructural domain is cloned into the carrier (pEU15.1) that contains people's heavy chain constant domain and regulatory element with the IgG heavy chain of the expressed in mammalian cell.Similarly, with V
LStructural domain is cloned into the carrier (pEU4.4) of expression people's light chain (λ) constant domain and regulatory element with the IgG light chain of the expressed in mammalian cell.Persic etc., 1997 have at first described the carrier of expressing heavy chain and light chain.Can be by introducing the simply carrier of engineered cambridge antibody technology company (Cambridge Antibody Technology) of OriP element.For obtaining IgG, heavy chain and light chain IgG expression vector are transfected into the EBNA-HEK293 mammalian cell.IgG enters substratum through expression-secretion.Merge gleanings, purifying after filtering.Adopt A protein chromatographic IgG purification.Medium supernatant is loaded on the sizeable A albumen ceramics pole (biological spectrum company (BioSepra)), with 50mMTris-HCl pH 8.0,250mM NaCl washing.Utilize 0.1M Trisodium Citrate (pH 3.0) in connection with IgG wash-out from the post, add Tris-HCl (pH 9.0) neutralization.Utilize ap10 post (peace agate West Asia company (Amersham), 17-0854-02 number) damping fluid of eluted material is replaced to PBS, utilization is based on the optical extinction coefficient of the aminoacid sequence of IgG, by the concentration (Mach etc. 1992) of spectrophotometry IgG.Adopt SEC-HPLC and SDS-PAGE to analyze cohesion or the degraded of IgG purification.
1.6 IL-17 induces IL-6 to discharge from the HT1080 cell
For measuring the biologic activity of IL-17 inhibitor, adopt HT1080 human desmocyte sarcoma cell test assessment scFv and IgG active.These cells react to human il-17 in the mode of dose-dependently and discharge IL-6.The details of test method is referring to " test materials and method " chapters and sections.
In this test, it is active (by their IC to the inhibition that 1nM people or 1nM stump-tailed macaque (cyno) non-human primates IL-17A react to measure one group of anti--IL-17scFv/IgG
50PH-value determination pH).The scFv that obtains and IgG render a service and are shown in table 4.
The effectiveness of scFv and IgG in the table 4:HT1080 test
NT does not test
The IA non-activity
In the HT1080 test, a little less than the scFV of leading separation suppresses usually, thereby fail to measure and render a service.Therefore, will show that inhibiting any scFv changes over IgG1 in the HT1080 test, in this test, again check to measure more accurately and render a service, thus can be to clone's rank.
Embodiment 2: antibody optimization
2.1 affinity maturation
Adopt directed mutagenesis method and avidity phage display to select to optimize leading antibody.Adopt described standard molecular biological technique (Clackson 2004), by the variable heavy chain (V of oligonucleotide guidance
H) and light chain (V
L) complementary determining region 3 (CDR3) mutagenesis produces the large library of scFv-phage derived from leading clone.These libraries are carried out selecting the variant higher to the avidity of IL-17A based on the phage display of avidity.Therefore, these variants can show the active raising of the inhibition of IL-17A and its receptors bind.Basically as previously mentioned (Thompson 1996) are selected.In brief, with scFv-phage particle and restructuring biotinylation human il-17 A incubation (bio-huIL-17A, inner HEKEBNA derive with interior finishing) in solution.Then according to manufacturer's recommendation with the coated paramagnetic bead of Streptavidin (
M-280) catch the scFv-phage of being combined with antigen.Then as previously mentioned the scFv-phage particle of (Osbourn 1996) rescue selection repeats this chosen process in the presence of the bio-huIL-17A that has concentration gradually to fall (being reduced to 10pM from 100nM in 5 take turns).Each clone of representative quantity of preparation selection result contains scFv pericentral siphon crude extract, basically screens as described in Example 1 them and suppresses the ability that people bio-huIL-17A is combined with human il-17 acceptor A.Thing is hit in screening, namely compare with representative parental generation antibody, show that the scFv variant that restraining effect obviously improves carries out dna sequencing, the variant of uniqueness is prepared into purifying scFv with further sign (referring to embodiment 1).
Then select some scFv, be transformed into as described in Example 1 IgG1 and again check.For extra increasing renderd a service, restructuring has produced the V that improves in the transfection step
HAnd V
L, the IgG1 that assessment obtains.It is believed that, be some uncertain technology at transfection level restructuring VH and VL, can successfully not produce and render a service the antibody that improves.Yet in this example, this technology is accidental successfully to have produced many efficient IgG, comprises antibody 3,4,5,7,9,10 and 12, as described below.
2.2 plant systemization
With optimize anti--V of IL-17A antibody
HAnd V
LKnown ethnic group in the aminoacid sequence of structural domain and the VBASE database is sequence alignment (Tomlinson 1997), identifies that by sequence similarity immediate kind is.V for Tina12 antibody pedigree
HStructural domain is VH3-23.For the VL structural domain, be V λ 6a.
Do not consider the Vernier residue (Foote and Winter 1992) not doing to change, V
H1 change is arranged, V in the framework of structural domain
L2 changes are arranged in the structural domain, adopt the standard side-directed mutagenesis, with suitable mutagenic primer all these changes being reverted back to immediate kind is that sequence is with identity ground match people antibody.Vernier residue in the VH structural domain is the terminal Arg of the C-among the VH FR3 (residue in the VH structural domain sequence number 98), and it does not change over kind is Lys.Vernier residue in the VL structural domain is the Ile (residue in the VL structural domain sequence number 47) among the VL FR2, and it does not revert back to kind is Thr, and the Phe among the VL FR2 (residue in the VL structural domain sequence number 50), and it does not revert back to kind is Tyr.
2.3 optimize the clone's
The receptor-ligand test
Adopt
Receptor-ligand is in conjunction with the restraining effect of in the selection result of experiment sieving optimization the human il-17 A (inner HEK EBNA derives) of HIS label being combined with IL-17RA Fc fusion rotein, as described in test materials and method chapters and sections.
Then adopt
Receptor-ligand is accredited as the effectiveness that the purifying scFv of thing is hit in screening in conjunction with test determination.The example that the leading scFv that obtains renders a service is shown in table 5.Data shown in this article are non-kind systemization antibody sequences.
The example that the leading scFv that optimizes in the test of table 5:HT1080 receptor-ligand renders a service
*The ND=undetermined is not because obtain these clones' scFv.Produce these clones by recombinating in the IgG phase.
2.4 optimize clone's HT1080 IL-6 release test
The leading antibody that separates of assessment is being optimized the active improvement situation of artifact in the HT1080 human fibrosarcoma cell described in " test materials and method " chapters and sections.Utilization is derived from the human il-17 A of HEK-EBNA expression system, carries out all tests derived from the stump-tailed macaque IL-17A of intestinal bacteria or HEK EBNA expression system.
One group of assessment is optimized in the HT1080 test that 1nM people or 1nM stump-tailed macaque (cyno) IL-17A is reacted in the dose-dependently mode anti--IL-17 scFv/IgG.The exemplary scFv that obtains and IgG render a service and are shown in table 6A.
ScFv and IgG render a service the example that improves in the table 6A:HT1080 test
*Further experiment (data set n=15) shows that geometric mean IC50 value is 0.8,95% fiducial interval 0.6-1.0.
2.5 detect the HT1080 test cell line that collaborative IL-6 discharges
The HT1080 cell can react and the collaborative IL-6 of release to human il-17 A and human TNF alpha in the concentration dependent mode.
Adopt antibody 2 that the improved form assessment of aforementioned HT1080 test cell line method optimizes and the biologic activity of 7 pairs of collaborative IL-6 reactions, wherein detect the IL-6 that 125pM human il-17 A and 25pM human TNF alpha are reacted and discharge (referring to " test materials and method " chapters and sections).
Antibody 2 and 7 all neutralizes and works in coordination with the IL-6 reaction.
Exemplary IC
50Value is shown in table 6b.
The example that IgG renders a service in the table 6b:IL-17A/TNF α HT1080 test
| Clone's title | IgG IC 50(nM) |
| Antibody 7 | 0.24(95％CI 0.19-0.28) |
| Antibody 2 | 0.41 |
2.6 suppress the reaction that natural IL-17A induces in the HT1080 cell
Utilization is derived from the ability of the natural origin of former generation human T-cell's IL-17A assessment antibody suppression IL-17A.The glycosylation site that has the N-connection from natural origin is different, the not glycosylation of restructuring IL-17A that intestinal bacteria derive.Utilize the cell-derived IL-17A assessment of T antibody can guarantee to keep the activity of utilizing protein for natural.
Adopt the T cell of the conditioned stimulus Healthy People donor that strengthens the IL-17A generation.These supernatant liquors contain other cytokine, for example can with the synergistic TNF α of any IL-17A that exists.Be with or without in the presence of the antibody, with the conditioned culture media adding HT1080 cell of T cell, and the above IL-6 of assessment baseline induces situation.Calculate thus IC
50Value.The method detailed of this test is seen test materials and method chapters and sections.
The effect that antibody induces IL-6 to produce to T cell conditioned medium liquid in the table 6c:HT1080 cell
(IC
50Value is calculated as nM, 95% fiducial interval)
2.7
Optimize selectivity and the species cross reactivity of antibody in the epi-position competition experiments
Adopt
The epi-position competition experiments, by detect to HIS label IL-17A (inner HEKEBNA derives) and various optimizations anti--restraining effect of IL-17 antibodies to be to determine that leading antibody is to selectivity and the species cross reactivity of IL-17 family.
In each test, test the titre of unlabelled Purification of Human IL-17A (inner intestinal bacteria derive), IL-17B (Pai Puluo technology company), IL-17C (RD system house), IL-17D (Pai Puluo technology company), IL-17E (RD system house) and IL-17F (Pai Puluo technology company), such as IC in the test
50Value is surveyed.
The titre (comprising people, stump-tailed macaque, dog (all being that inner intestinal bacteria derive) and Muridae IL-17A (RD system house)) of the various IL-17 of test is to determine to optimize the species cross reactivity of antibody in each test.
The results are summarized in table 7.Raw data shows that the preamble optimization antibody of test is selective for human il-17 A, nonrecognition family member IL-17B, C, D, E and F.
In another embodiment, when using with 1 μ M or higher concentration, observing 7 couples of human il-17 F of antibody has part to suppress.
In addition, the effectiveness degree of all antibody recognition people, stump-tailed macaque and the dog IL-17 of test is different.The equal nonrecognition Muridae of the antibody of any optimization IL-17A.
Table 7: human il-17 A is in conjunction with the IgG test of optimizing
The effectiveness of middle IL-17 family member and different I L-17 kind
B=human il-17 B
C=human il-17 C
D=human il-17 D
E=human il-17 E
F=human il-17 F
The NI=unrestraint
ND=does not detect
*2 times of calculating subsequently of numeric representation in the bracket or the average IC of independent experiment repeatedly
50Value.
Embodiment 3: the avidity of antibody on human IL-17A
3.1 pA
2Analyze
For further characterizing anti--IL-17A antibody, adopt test materials and the described method antagonist of method chapters and sections to carry out pA
2Analyze.This is analyzed for assessment of the equilibrium dissociation constant (Kd) of IgG to human il-17 A.The human il-17 A that utilizes HT1080 human fibrosarcoma cell and HEK EBNA to express analyzes.
Analyze to estimate anti--IL-17 IgG to the avidity of human il-17 A between 65pM and 550pM, example data sees Table 8.Also analyze with the mAb of biogenic company and negative control mAb.The avidity of separation antibody shows higher 2 and 5 times than the mAb of biogenic company.
Table 8a:IgG is to the exemplary avidity of human il-17 A
| pA 2Value | Kd(pM) | |
| Antibody 2 | 9.73 | 188 |
| Antibody 7 | 9.23 | 553 |
| The mAb of biogenic company | 8.96 | 1100 |
3.2 utilize the binding affinity data of biosensor analysis
Adopt BIAcore 2000 (GE keep healthy company (GE Healthcare)) to assess the kinetic parameter of the IL-17A interaction of antibody 7 and different plant species.
Surface concn due to the analyte molecule that biosensor utilizes the optical effect research of surperficial plasmon resonance (SPR) to flow through on ligand molecular interacts changes, and described ligand molecular links to each other with the dextran layer covalency of biologic sensor chip.The analyte of limiting concentration is flow through on the part of coupling, detect in conjunction with situation, for example local spr signal increases (in conjunction with the phase).Damping fluid is flow through, can observe during this period analyte and dissociate, for example signal reduces (dissociating the phase).Then the part of all the other analytes and chips incorporate can be peeled off, repeat this process with several different analyte concentrations.Experimental session utilizes a series of contrasts can obviously not change in whole experimentation with absolute binding ability or the kinetics distribution situation of guaranteeing the coupling part usually.Special-purpose Hepes buffer saline (HBS-EP) be typically used as the main dilution buffer liquid of analyte sample and dissociate during mobile damping fluid.Be (second) any resonance units of becoming (RU is with the direct corresponding arbitrary unit of spr signal) in time with Experiment Data Records.The specific refractory power of RU and chip surface is directly proportional, so it is the suitable tolerance of the analyte quality of combination.Then can utilize BIA assessment software bag processing data, according to data set match combination model.Association (the M that returns
-1s
-1) and (s that dissociates
-1) velocity constant can calculate association (M
-1) and (M) affinity constant that dissociates.
Adopt the avidity of single experiment calculation antibody 7, wherein IgG
1With special-purpose CM3 chip surface covalency (amine is connected) coupling, final RU surface density is 300.Then, so that a series of diluents (0.78-50nM) of recombinant human or stump-tailed macaque IL-17A flow through antibody 7 successively.The molarity of part adopts the method for publishing to calculate optical extinction coefficient according to the 280nm absorbancy.From IgG
1The blank wandering cells data of middle deduction are from the blank damping fluid of general data collection deduction HBS-EP (" dual blank deduction ").Then utilize simultaneously BIA to assess 3.2 softwares according to 1: 1 Lang Gemiuer model of the data fitting of each analyte concentration collection and divalence analyte model.
According to the κ 2 that calculates and the validity of T value (parameter value/deviation value) assessment/bound data, the two must distinguish＜2 and＞100.
Antibody 7 is fixed in the surface of CM3 chip, makes a series of diluents (0.78-50nM) of restructuring IL-17 variant flow through successively IgG
1Data are fitted to 1: 1 Lang Gemiuer model (while k simultaneously
ak
d) and divalence analyte model (simultaneously ka kd).Visual observations and they are better than κ
2Value judges that the model-fitting of divalence analyte gets better.Then the ratio kd1/ka1 from velocity constant calculates affinity constant (Kd).
Table 8b: the dynamic analysis of antibody 7
| Antigen | 1: 1 Lang Gemiuer K D (nM) | Divalence analyte K D (nM) |
| Human il-17 A (intestinal bacteria) | 0.195 | 2.098 |
| Cyno IL-17A (intestinal bacteria) | 0.512 | 1.340 |
| Human il-17 A-label-His10 (HEK) | 0.171 | 0.41-1.88 |
Embodiment 4: suppress the IL-17A induced activity in the former generation chondrocyte of people
Destroying joint cartilage is the feature that comprises many inflammatory diseasess of rheumatoid arthritis and osteoarthritis.Main cell type is the chondrocyte in the joint cartilage.Can be from the former generation chondrocyte of separate tissue people of total knee replacement intra-operative taking-up.By the cell of collagenase digesting extraction cartilage, cultivate in vitro tests under the specified conditions.When stimulating the chondrocyte with restructuring or natural IL-17, it produces a large amount of inflammatories and destructive medium, comprises cytokine (for example IL-6, IL-8), PGE2 and degrading enzyme, for example MMP13.The test materials of this paper and method chapters and sections are described this test in detail.
Table 9a and 10a show the master data of the IgG1 antibody that is used for this test.Table 9b and 10b are shown as the more new data that larger data collection (increase of n numerical value) calculates.
Table 9a: the master data that anti--IL-6 that IL-17A antibody suppression IL-17A induces discharges from the former generation chondrocyte of people
| IgG1 | 0.2nM IL-17A | n | 2nM IL-17A | n |
| FORD023A05 | 2.77(1.05-7.31) | 4 | 5.75(2.69-12.30) | 4 |
| Antibody 2 is (non--kind of being *) | 0.85(0.44-1.65) | 4 | 5.76(3.39-9.90) | 4 |
| The mAb of biogenic company | 1.02(0.78-1.32) | 9 | 5.18(4.02-6.67) | 12 |
| TINA10 | 27.23(10.02-74.00) | 5 | 87.97(52.92-146.24) | 7 |
| Antibody 1 | 29.85(11.23-79.29) | 4 | 75.67(27.55-207.82) | 4 |
Geometric mean IC
50Represent (95% confidence limit) with nM.
*Antibody 2 in this test with the utilization of non-being of kind form.
Table 9b: the more new data that anti--IL-6 that IL-17A antibody suppression IL-17A induces discharges from the former generation chondrocyte of people
| IgG1 | 0.2nM IL-17A | n | 2nM IL-17A | n |
| Antibody 2 | 1.52(0.61-3.78) | 5 | 5.51(3.54-8.57) | 5 |
| Biogenic mAb | 1.03(0.80-1.33) | 12 | 5.70(4.64-7.01) | 12 |
| Antibody 7 | 0.51(0.18-1.43) | 6 | 1.44(0.79-2.64) | 6 |
Geometric mean IC
50Represent (95% confidence limit) with nM.
Table 10a: the master data that anti--IL-8 that IL-17A antibody suppression IL-17A induces discharges from the former generation chondrocyte of people
| IgG1 | 0.2nM IL-17A | n | 2nM IL-17A | n |
| FORD023A05 | 2.77(1.86-4.11) | 7 | 7.84(4.12-14.90) | 7 |
| Antibody 2 is (non--kind of being *) | 0.74(0.46-1.18) | 7 | 3.81(2.24-6.45) | 7 |
| Antibody 2 | 2.30 | 3 | 7.91 | 3 |
| Antibody 7 | 0.17 | 3 | 1.60 | 3 |
| Biogenic mAb | 0.78(0.52-1.17) | 10 | 3.98(3.09-5.12) | 14 |
| TINA10 | 21.42(12.85-35.69) | 7 | 61.96(45.76-83.90) | 9 |
| Antibody 1 | 18.12(10.7-30.67) | 4 | 44.47(39.93-49.53) | 4 |
Geometric mean IC
50Represent (95% confidence limit) with nM.
*Antibody 2 in this test with kind of being and non-kind systemization formal testing.
Table 10b: the more new data that anti--IL-8 that IL-17A antibody suppression IL-17A induces discharges from the former generation chondrocyte of people
| IgG1 | 0.2nM IL-17A | n | 2nM IL-17A | n |
| Antibody 2 | 1.49(0.80-2.78) | 8 | 7.18(4.38-11.75) | 8 |
| Antibody 7 | 0.30(0.14-0.63) | 8 | 1.30(1.10-1.54) | 9 |
| Biogenic mAb | 0.85(0.58-1.24) | 14 | 5.09(4.06-6.37) | 15 |
Geometric mean IC
50Represent (95% confidence limit) with nM.
Table 11: anti--MMP13 that IL-17A antibody suppression IL-17A induces discharges from the former generation chondrocyte of people
| IgG1 | 0.2nM IL-17A | n | 2nM IL-17A | n |
| Antibody 2 | 1.49(0.80-2.78) | 8 | 7.18(4.38-11.75) | 8 |
| Antibody 7 | 0.30(0.14-0.63) | 8 | 1.30(1.10-1.54) | 9 |
| Biogenic mAb | 0.85(0.58-1.24) | 14 | 5.09(4.06-6.37) | 15 |
Geometric mean IC
50Represent (95% confidence limit) with nM.
*Antibody 2 in this test with the utilization of non-being of kind form.
Table 12: anti--PGE that IL-17A antibody suppression IL-17A induces
2Discharge from the former generation chondrocyte of people
| IgG1 | 2nM IL-17A | n |
| FORD023A05 | 4.75 | 3 |
| Antibody 2 (non-kind systemization *) | 1.94 | 3 |
| Biogenic mAb | 2.83 | 3 |
| TINA10 | NT | |
| Antibody 1 | NT |
Geometric mean IC
50Represent (95% confidence limit) with nM.
NT: not test
*Antibody 2 in this test with the utilization of non-being of kind form.
Embodiment 5: suppress the Biological acdtivity in vivo that IL-17A induces in the air bag of Balb C mouse
5.1 air bag forms
With isoflurane anesthesia Balb C mouse, scrape off the hair of back surfaces.Be to form air bag, with the 25g syringe needle with the subcutaneous injection of 2.5ml sterile air in the back midline.After clear-headed mouse is transferred back to their cage house, held onto carefully mouse at the 3rd day, with the 2.5ml sterile air air bag is reexpanded.
Adopt two kinds of method assessment antibody (IgG1): premixed, the method is mixed antibody first again injection with IL-17, and general, the method gives first antibody and injects IL-17A again.In the situation that general gives, with aseptic PBS dilution antibody, the 5th day as bolus through peritoneal injection.
Again held onto carefully mouse at the 6th day, give separately injection (giving in the situation of mouse in contrast or general) in each mouse capsule or contain 1ml volume 0.5% methylcellulose gum of the human il-17 A (interior reagent) that intestinal bacteria derive with anti--IL-17 antibody combination injection (being pre-mixed antibody and IL-17A).In these experiments, antibody and part are mixed, be injected into again air bag in 30 minutes 37 ℃ of preincubation first.Mouse is transferred back to their cage house until finally take a sample the CO that raise with concentration this moment
2Select and kill mouse.For helping to reclaim the elutant of air bag, the 2ml PBS that will contain 4mM EDTA carefully is injected into this air bag, about 15 seconds of this air bag of applying light.From air bag, take out carefully elutant, pour into graduated polypropylene test tube.The volume that record reclaims.With sample be maintained at preserve on ice to be processed.
5.2 elutant analysis
Total leukocyte (TWBC) counting
In each the Coulter tank that contains 10ml Isoton, add 20 μ l elutants.To sample double reading, obtaining the double counting of each sample with Coulter Z1 counter, background reading maintains＜and 2000.Then the elutant volume of considering to reclaim from air bag is to regulate cell count.
The IL-6 quantitative assay
With the centrifugal sample of 1500rpm 5 minutes, with 300 μ l supernatant liquor equal portions with triplicate adding 96 orifice plates, be stored in-20 ℃ to be analyzed.According to the test kit scheme, utilize the Quantikine of RD system house mouse IL-6 cytokine ELISA test kit (catalog number (Cat.No.) M6000B) to analyze 50 μ l standard substance, contrast and elutant sample.
Utilize 450nm and tuning wavelength to be the dull and stereotyped reader of the SoftMax Pro ELISA of 540nm, the concentration of IL-6 from mouse IL-6 standard curve determination elutant.
5.3 IL-17A induces IL-6 to discharge in the inhibition air bag
Table 13a: IL-17A induces IL-6 to discharge in the inhibition mouse air bag: the antibody that is pre-mixed and IL-17A
*Antibody 2 in this test with the utilization of non-being of kind form.
Table 13b: IL-17A induces IL-6 to discharge in the inhibition mouse air bag: general gives antibody
5.4 IL-17A induces the white corpuscle flux in the inhibition air bag
Table 14a: IL-17A induces the white corpuscle flux in the inhibition air bag: the antibody that is pre-mixed and IL-17A
*Antibody 2 in this test with the utilization of non-being of kind form.
Table 14b: IL-17A induces the white corpuscle flux in the inhibition air bag: general gives antibody
Embodiment 6:IL-17A/TNF α Synergism Testing
6.1 after death the IL-17A/TNF α in the cartilage explant acts synergistically
Destroying joint cartilage is the feature that comprises many inflammatory diseasess of rheumatoid arthritis and osteoarthritis.Main cell type is the chondrocyte in the joint cartilage.Can be from total knee replacement intra-operative or the separate tissue human cartilage that takes out from donor after death.Downcut the outer planting body disc from cartilage, cultivate in vitro tests under aseptic condition, the chondrocyte is retained in their cell matrix.When stimulating explant with IL-17 or TNF α, it produces a large amount of inflammatories and destructive medium, comprises cytokine, for example IL-6.Test materials and method chapters and sections provide the method detailed of this test.
In assessment IL-17/TNF α Synergism Testing, in the preliminary experiment of the functional effect of antibody in the cartilage explant, do not obtain IC
50Data are because tissue supply is limited.When calculating does not have the set member (0% suppresses), the % of the binding members of given concentration suppresses and reaction.Data are shown in table 15a.In other experiment, the IC50 data have been calculated, shown in table 15b.
Collaborative IL-6 reaction is induced in IL-17A and TNF α combination, and the IL-17A neutralizing antibody suppresses this reaction.The isotype control antibodies does not affect this concerted reaction.
Table 15a: antibody is after death worked in coordination with the effect (standard deviation of error=12 time repetition, n=1 position donor) that IL-6 reacts to IL-17A and TNF α in the cartilage explant the people
*Antibody 2 in this test with the utilization of non-being of kind form.
Table 15b: antibody is after death worked in coordination with the IC that IL-6 reacts to IL-17A and TNF α in the cartilage explant the people
50(average data, 95% fiducial interval)
| Antibody (IgG1) | Average IC 50(95％CI) | n |
| Antibody 2 | 3.39 | 2 |
| Antibody 7 | 0.61(0.26-1.42) | 5 |
6.2 the synergy of the IL-17A/TNF α in the OA synovioblast
The feature of joint injury comprises the synovial membrane that has inflammation in such as diseases such as rheumatoid arthritiss, and it is by immune cellularity, for example the inoblast of scavenger cell, T cell, B cell and propagation.The synovial membrane that can separate from the joint tissue of obtaining at the total knee replacement intra-operative inflammation.The collagenase digesting synovial membrane, the cell of culture of isolated is used in vitro tests under aseptic condition, and wherein chief component is inoblast.When stimulating inoblast with IL-17 or TNF α, it produces a large amount of inflammatories and destructive medium, comprises cytokine, for example IL-8.Test materials and method chapters and sections provide the method detailed of this test.
Adopt two groups of different cytokine concentration, IL-17A and TNF α induce collaborative IL-8 reaction together.Utilize the IL-17 neutralizing antibody can reduce this reaction, although the isotype control antibodies to concerted reaction without effect.
Table 16a: (cytokine concentration is in pg/ml to the effect of reacting with the collaborative IL-8 of IL-17A (10ng/ml) and TNF α (1ng/ml) in people OA synovioblast for antibody, the standard deviation of error=triplicate data point, n=1 position donor)
| IL-8(pg/ml) | ||
| Antibody (IgG1) | Mean value | % suppresses |
| IL-17A 10ng/ml+ TNF 1ng/ml | 62519.10 | 0 |
| The contrast of IL-17A+TNF+ hIgG1 isotype | 64967.81 | -3.9 |
| IL-17A+TNF+ FORD23A05 | 32117.64 | 48.6 |
| IL-17A+TNF+ antibody 2 | 23340.13 | 62.7 |
| IL-17A+TNF+ antibody 7 | 20829.47 | 66.7 |
Table 16b: (cytokine concentration is in pg/ml to the effect of reacting with the collaborative IL-8 of IL-17A (1ng/ml) and TNF α (0.01ng/ml) in people OA synovioblast for antibody, the standard deviation of error=triplicate data point, n=1 position donor)
| IL-8(pg/ml) | ||
| Antibody (IgG1) | Mean value | % suppresses |
| IL-17A 1ng/ml+ TNF 0.01ng/ml | 3255.86 | 0 |
| The contrast of IL-17A+TNF+ hIgG1 isotype | 3298.59 | -1.3 |
| IL-17A+TNF+ FORD023A05 | 1731.59 | 46.8 |
| IL-17A+TNF+ antibody 2 | 1506.59 | 53.7 |
| IL-17A+TNF+ antibody 7 | 1107.75 | 66.0 |
6.3 IL-17A and IL-17A/TNF α induce IL-8 in the inhibition RA synovioblast
As described in 6.2, the synovial membrane of inflammation is the key feature of RA.Also can adopt the joint tissue that during the RA joint replacement surgery, takes out with described identical method and separate the RA synovial membrane.These cells are relevant with disease, and IL-17A and TNF α are reacted and produce the cytokine that comprises IL-8.When adding two kinds of cytokines simultaneously, produce concerted reaction.Test materials and method chapters and sections provide the method detailed of this test.
Table 17: the effect (IC of the IL-8 reaction that antibody is induced IL-17A/TNF α in people RA synovioblast
50) (average data, 95% fiducial interval)
| Antibody (IgG1) and stimulation | Average IC50nM, 95%CI | n |
| Antibody 7 and IL-17A (2nM) | 2.85(1.96-4.15) | 3 |
| Antibody 7 and IL-17A (1nM) and TNF α (0.1ng/ml) | 4.97(1.48-16.73) | 3 |
Embodiment 7: the epitope mapping that antibody 7 is combined with human il-17 A
7.1 antibody 7 disturbs the H/D exchange velocity of IL-17A
In the first experiment, make the D in the IL-17A exchange solution
2O is with itself and antibody 7 (IgG1) combination that is fixed on the post, then at H
2Reply exchange among the O and still be combined with antibody column simultaneously, thereby cause epi-position replying permutoid reaction and be protected with during deuterium-labeled subsequently.In the second experiment, first IL-17A is incorporated into the antibody 7 (IgG1) on the post, then use D
2The O mark exchanges back H at last
2O still is combined with antibody column simultaneously, thus IL-17A without a part by deuterium-labeled.Then measure the difference of peptide quality between two experiments.The difference of deuterate level is the tolerance that exchange lags behind during with antibodies between two experiments.The materials and methods chapters and sections provide the method detailed of this scheme.
The end-labelled IL-17A of this research and utilization N-, its sequence is shown in Fig. 1 (SEQ ID NO:197).Residue 1-21 be expressed as testing goal and add peptide-labeled, comprise " Avitag
TM", thereby can carry out at the lysine residue place of mark specifically enzymatic living beings elementization.Gly22 is the first residue of ripe IL-17A.
Show that unique zone that the H/D exchange velocity is subjected to obviously to disturb is between the amino acid 92-108 of SEQ ID NO:197 shown in Figure 1, comprise sequence C RHLGCINADGNVDYHM (SEQ ID NO:199).This sequence is corresponding to the residue 71-87 of ripe human il-17 A (SEQ ID NO:198).
Table 18: relatively the deuterate during binding antibody 7 and when exchange back proton and binding antibody in solution deuterate and exchange back the deuterate level difference per-cent of proton.Particularly 10% of time point above difference is regarded as significantly in early days.The residue numbering of sequence is seen Fig. 1 (SEQ ID NO:197).
7.2 analyze mutant IL-17A and antibody 7 in conjunction with situation
Take alternative plan to confirm that the epi-position of identifying among the embodiment 7.1 is correct.This scheme is utilized following observations, and namely antibody is not combined with Muridae IL-17A, although Muridae IL-17A can signal to induce IL-6 synthetic by human il-17 A acceptor.The peptide sequence (CRHLGCINADGNVDYHM, SEQ ID NO:199) of identifying among the embodiment 7.1 has 7 places and changes between people and Muridae sequence.If should really be included in the epi-position of 7 combinations of antibody in the zone, then when all these 7 residues change over Muridae residue of equal value from people's residue, estimate that the mutant IL-17A that obtains also can work with people's acceptor, but can be again by antibody 7 combinations or inhibition.
Mutant IL-17A has following change (L74Q, G75R, I77V, D80E, N82K, V83L, Y85H), as shown in Figure 2 (SEQ ID NO:200).After removing signal sequence, the first residue is Gly24.Add 5 histidine residues to assist purifying at the C-end.The mature form of mutant human IL-17A is SEQ ID NO:201.
Therefore, clone and expression mutant IL-17A in Mammals HEK/EBNA clone.Collect substratum, adopt Ni chromatography purification protein.The materials and methods chapters and sections are described this scheme in detail.By identical scheme clone wild-type IL-17A, then express and purifying.
According to manufacturer's working instructions, adopt BIAcore analyze wild-type and mutant IL-17A molecule and the antibody 7 (IgG1) that is fixed in the CM5 chip in conjunction with situation.This scheme causes being fixed as 5275 reactons (RU).
The antibody 7 that table 19:IL-17A wild-type and mutant and CM5 sensor chip surface are fixed in conjunction with situation
| Part | Average RU (+SEM) |
| Mutant IL-17A | 8.0(5.4) |
| Wild-type IL-17A | 539.1(0.2) |
With the contrast IgG (cambridge antibody technology company) without combination.Wild-type IL-17A and the antibody 7 strong combinations of fixing.On the contrary, mutant IL-17A shows not and antibody 7 combinations.Observe RU signal low be attributable to only to have powerful connections " noise ".Mutant IL-17A does not have detectable combination to confirm that further sequence shown in the embodiment 7.1 (SEQ ID NO:199) does not contain at least a portion of antibody 7 epi-position of identifying, i.e. one or more residues in the antibodies SEQ ID NO:199.
7.3 the effect that antibody induces IL-6 to discharge from the HT1080 cell to mutant IL-17A
For measuring the biologic activity of mutant IL-17A (embodiment 7.2 described mutant), assessment reaction in HT1080 human fibrosarcoma cell test.The details of test method can be referring to " test materials and method " chapters and sections.
In this test, the EC that assessment IL-17A (intestinal bacteria, Pai Puluo technology company or HEK EBNA derive in inside) or mutant IL-17A (inside, HEK EBNA derives) discharge IL-6
50(obtaining the concentration of half highest response) (table 20a).The second, measure the inhibition active (by their IC50 pH-value determination pH) that anti--IL-17 IgG (the highest 50nM) reacts to IL-17A or mutant IL-17A (utilizing inferior peak concentration).For example, table 20b shows that antibody 7 (for example IgG1) is to the effectiveness of various forms IL-17A.Not proof contrast IgG inhibition IL-6 reaction in this test.
The exemplary EC that IL-17A or mutant IL-17A induce IL-6 to discharge in the table 20a:HT1080 test
50
| Part | The source | EC50(nM) |
| IL-17A | Pai Puluo technology company (intestinal bacteria) | 0.40 |
| IL-17A | Inner (HEK EBNA) | 0.52 |
| Mutant IL-17A | Inner (HEK EBNA) | 2.36 |
The exemplary IC of 7 couples of IL-17A of antibody or mutant IL-17A in the table 20b:HT1080 test
50
| Part | The source | [part] (nM) | Antibody 7 IC 50(nM) |
| IL-17A | Pai Puluo technology company (intestinal bacteria) | 1 | 0.35 |
| IL-17A | Inner (HEK EBNA) | 2 | 0.43 |
| Mutant IL-17A | Inner (HEK EBNA) | 1 | Unrestraint |
7.4 the X-ray crystal structure of IL-17A antibody complex is measured
Measure the structure of the IL-17A of being combined with antibody 7Fab by the X-radiocrystallography.The materials and methods chapters and sections have been listed Fab generation, the preparation of IL-17A albumen, crystallization and crystallographic method.
Obtain the crystal of IL-17A/Fab mixture, it is oblique crystal spacer P2
1(monoclinic spacegroup P2
1) laminar crystal.Obtain 2.6
The complete diffraction data of resolving power.Utilize the Fab fragment as trial model, differentiate structure by molecular replacement, thus the Fab fragment is directed and be positioned in the crystallization asymmetric cell.4 Fab fragments are arranged in the asymmetric cell.The IL-17A dimer can be simulated other electron density.In asymmetric cell, find two IL-17A dimers.
In crystalline structure, each IL-17A dimer is in conjunction with two Fab fragments.The IL-17A dimer is a kind of prolongation molecule take long β chain as feature.This similar is in (Hymowitz etc., 2001) that IL-17F reports.The IL-17A dimer is sandwich between two Fab fragments.Two interaction sites are of equal value, and they are correlated with by IL-17A dimer symmetry.The IL-17A dimer is unordered away from the part in conjunction with the Fab fragment, and namely it is pliable and tough and takes different orientations in whole crystal, thereby makes electron density reach balance.Therefore, can not make up the model of dimeric this part of IL-17A.Therefore, this mixture only produces and interacts by the numerator mediated lattice of Fab.For utilizing (2001) the used specifiers such as Hymowitz, the epi-position interaction sites is near " neckline " of structure, and " skirt " zone of opposite end is unordered.
Crystalline structure can interact with the epi-position between atom detail detection IL-17A and the Fab.Shown in table 22.
Table 22:IL-17A/Fab direct interaction
In table 22, residue number comprises chain designator (H:Fab heavy chain, L:Fab light chain, the monomer A among the A:IL-17A, the monomers B among the B:IL-17A).The residue numbering of IL-17A is corresponding to mature sequence SEQ ID NO:198.The residue numbering of Fab VH and VL structural domain corresponds respectively to SEQ ID NO:62 and SEQ ID NO:176.The Kabat residue numbering that has also shown Fab.
*Asterisk represents Fab framework residue.What be used for hydrogen bond is 3.2 apart from cutoff value
Apolar interaction be 4.0
Adopt CCP4 program CONTACT (CCP4,1994) to obtain distance.Only need to describe one of two interaction sites, because they are of equal value.Interaction relates to the amino-acid residue of two monomers in the heavy chain of antibody fragment and the complementary determining region of light chain (CDR) and the IL-17A dimer.A in heavy chain of antibody and the IL-17A dimer and B chain interact, and light chain only interacts with monomers B.The amino-acid residue that consists of the interaction sites part among the IL-17A is Ser40-Tyr43 (comprising end points) and the Arg46 in the dimer A chain, with Leu74, Pro91, Tyr85-Asn88 and the Pro126-Ile127 in the dimer B chain.The residue that the Fab light chain provides is Ala30-Tyr33 (comprising end points), Phe50, Gln54, Tyr94 and Pro96.The residue of heavy chain is Thr28, Ser30-Tyr32, Tyr59 and L100-H102.Therefore, in IL-17A, 12 amino acid form the epi-position site, with 16 amino-acid residues interactions in the antibody.Interaction comprises 9 hydrogen bonds and nonpolar Van der Waals interaction (being also referred to as hydrophobic interaction).Some hydrophobic interactions comprise the mutual effect of electronics accumulative facies, relate to the pi electronics in the aroma system.
Detecting crystalline structure shows and it seems can replace with the more interactional Fab residues of IL-17 and keep interactional other residue.
For example, the hydrogen bonded between Ala30L (the Kabat residue 29 among the LCDR1) and the IL-17 does not relate to the Ala side chain that is positioned at the binding members surface in the main chain oxygen of L-Ala.This shows that other amino-acid residue can replace the Ala of this position, and does not lose this interaction, therefore, can not cause the avidity of binding members and IL-17A to reduce.
Hydrogen bond also can form between the backbone atoms of Ser31H, Leu100H, Ala30L and His102H place binding members, shows other residue that can replace these positions and does not lose these hydrogen bonds.
The main chain C-α of Asn31L (the Kabat residue 30 among the LCDR1) and IL-17 form apolar interaction, and replacing this residue can not affect yet.
According to structure detection, consider position and the character and sterically hindered of combination, below be other the non-limit example that shows with the interactional residue of IL-17:
Ser, Ala, Gly, Thr or the Cys at Kabat residue 31 places of-HCDR1
The Tyr at Kabat residue 32 places of-HCDR1
Tyr or the Phe at Kabat residue 58 places of-HCDR2
The hydrophobic residue at-Kabat residue 96 places, for example Leu, Ile, Val, Ala or Phe
The hydrophobic residue at Kabat residue 97 places of-HCDR3, for example Ile, Leu, Val, Ala or Phe
The ring-type residue at Kabat residue 98 places of-HCDR3, for example His, Trp or Phe
Tyr or the Phe at Kabat residue 31 places of-LCDR1
Tyr or the Phe at Kabat residue 32 places of-LCDR1
The polar residues at Kabat residue 53 places of-LCDR2, for example Gln
The Tyr at Kabat residue 91 places of-LCDR3
Pro, oxyproline, His, 3-Methyl histidine or the Asp at Kabat residue 93 places of-LCDR3
Thr or the Ser at Kabat residue 28 places among-the heavy chain FR1
Ser or the Thr at Kabat residue 30 places among-the heavy chain FR1
The hydrophobic residue at Kabat residue 49 places, for example Phe, Tyr or His among-the light chain FR2.
Test materials and method
HEK EBNA cell
HEK EBNA cell (ATCC catalog number (Cat.No.) CRL-10852) is available from hero company (Invitrogen).
IL-17 concentration
In all tests of following and its place's description of this paper, the concentration of IL-17A or IL-17 homologue refers to the concentration of the IL-17 homodimer that disulfide linkage connects.
The receptor-ligand combination
Test
With the receptor-ligand combination
(homogenizing time-resolution fluorescence) test method screening selection result is to the human il-17 A (inner HEK EBNA derives) of biotinylation human il-17 A (200-17 of Pai Puluo technology company) or the HIS label inhibition situation in conjunction with IL-17RA Fc fusion rotein (177-IR of RD system house).
Do not do dilution (leading separation) or dilution (preamble optimization) is screened the result, in 50mM MOPS pH of buffer 7.4, preparation contains the pericentral siphon crude extract of scFv in 0.5mM EDTA and the 0.5M sorbyl alcohol.Under the room temperature, with 3nM biotinylation human il-17 A (6nM monomer) or 3nM HIS label human il-17 A (6nM monomer) and 3.2nM Streptavidin cryptate compound (the biological 610SAKLB of international corporation of CIS) or 1.6nM with the cryptate compound mark anti--respectively lucifuge preincubation 1 hour of label IgG (the biological 61GF2KL of international corporation of CIS).All are diluted in the 50mM HEPES damping fluid (test damping fluid) that contains 0.4M Potassium monofluoride and 0.1%BSA carries out.Simultaneously, under the room temperature with 12nM IL-17RAFc fusion rotein with the lucifuge preincubation 1 hour in the test damping fluid of the 40nM Anti-Human Fc antibody (the biological 61HFCXLA of international corporation of CIS) of XL665 mark.
After the reagent preincubation, the rough scFv sample of 10 μ l is added the low volume test panel (Costar3676) in 384 holes.Add subsequently acceptor and the anti--Fc XL665 mixture of 5 μ l preincubation, then add 5 μ l human il-17 A and cryptate compound and detect mixture.
The final concentration of used IL-17A is 0.75nM in this test.
Then at room temperature with test panel with 1000rpm centrifugal 1 minute, incubation is 5 hours under the room temperature, uses the dull and stereotyped reader of EnVision (Pa Jin Elmer Co., Ltd) to read the time meta-resolution fluorescence of 620nm and 665nm emission wavelength again.
By calculating the % Δ F value analytical data of each sample.Measure Δ F according to equation 1.
Equation 1:
As described in equation 2, utilize % Δ F value to calculate the % specific binding subsequently.
Equation 2:
At two kinds
The purifying scFv of the positive colony that test screen is identified in the test is to the inhibition situation of IL-17A and IL-17 receptors bind.Adopt titration scFv concentration to determine the IC by test
50The clone of pH-value determination pH renders a service.Utilize 4-parameter logical equatiion (equation 3), adopt GraphPad Prism software to measure IC by fitting of a curve
50Value.
Equation 3:
At the bottom of the Y=+(top-end)/(1+10^ ((LogEC50-X) * HillSlope))
X is the logarithm of concentration.Y is specific binding.
Y starts from the bottom, rises on the top with S shape.
All tests comprise reference anti--IL-17mAb (biogenic company) is as positive control.
Suppress the epi-position competition of HIS label human il-17 A and Anti-Human IL-17A antibodies
Test
Can adopt this epi-position competition experiments to measure between binding members competition in conjunction with IL-17A.This test also can be measured binding members in conjunction with the IL-17 of different plant species, and for example cross reactivity of people and stump-tailed macaque IL-17A, and/or binding members is in conjunction with other IL-17 family member, for example cross reactivity of IL-17 homologue A-F.The binding members of testing in this test is the scFv form normally, tests to be applicable to IgG or other binding members form although the technician can improve this.The concentration of reagent usually with antibody to the avidity of IL-17A and different.
Under the room temperature, the 1.6nM of 2nM people HIS label IL-17A (4nM monomer) and cryptate compound mark is anti--label IgG lucifuge preincubation 1 hour.All are diluted in the test damping fluid carries out.Simultaneously, under the room temperature with 0.6nM anti--IL-17 IgG1 (will detect test binding members and its competition) with the lucifuge preincubation 1 hour in testing damping fluid of the 20nM Anti-Human Fc antibody of XL665 mark.
After the reagent preincubation, 10 μ l samples are added the low volume test panel in 384 holes, duplicate.Add subsequently HIS label human il-17 A and the anti--label cryptate compound mixture of 5 μ l preincubation, then add 5 μ l human il-17 antibody and XL665 and detect mixture.
Then at room temperature with test panel with 1000rpm centrifugal 1 minute, incubation is 2 hours under the room temperature, uses the dull and stereotyped reader of EnVision (Pa Jin Elmer Co., Ltd) to read the time meta-resolution fluorescence of 620nm and 665nm emission wavelength again.
By calculating the % Δ F value analytical data of each sample, utilize subsequently this % Δ F value to calculate the % specific binding.
Adopt GraphPad Prism software to measure IC
50Value.
The discussion of HTRF test usually can be referring to the receptor-ligand combination
The method of test.
HT1080 induces the IL-6 release test
With HT1080 cell (cell culture Europe preservation institute (European Collection of CellCultures), ECACC-ECACC number 85111505) with 5 * 10
4Cells/well is seeded in the flat tissue culture ware in 96-hole (Rodrigo Costa company).Then with cell in 200 μ l test mediums (minimum essential medium (MEM) that contains following component: Earles salt and L-glutaminate (hero company), the heat-inactivated foetal calf serum of 10% (v/v) (FBS) (hero company), 1% (v/v) be non-without the MEM-of L-glutaminate-indispensable amino acid (hero company)), with 37 ℃ and 5%CO
2Moistening atmosphere overnight incubation.
With the titrating solution of phosphate-buffered saline (PBS) preparation purifying scFv/IgG, 37 ℃ in test medium with IL-17A (1nM final concentration) preincubation 30-60 minute.Substratum is detached cell, add simultaneously 100 μ l/ hole test mediums.To wherein adding 100 μ l/ hole samples, (18 hours ± 4 hours) are incubated overnight.Collect supernatant liquor, check immediately or be stored in-20 ℃.
Utilize the IL-6 level in people IL-6 Duoset ELISA test kit (RD system house) the mensuration supernatant liquor.On with the coated FluoroNunc Maxisorb flat board that spends the night of 2 μ g/ml IL-6 capture antibodies (Ab) (with PBS dilution, 100 μ l/ holes), carry out ELISA.Wash ELISA dull and stereotyped three times with the PBS-tween, add 300 μ l reagent thinners (1% bovine serum albumin (BSA) of PBS preparation).The lower incubation of room temperature (RT) washs the ELISA flat board after 1 hour as previously mentioned.Between 1 hour incubation period, with 1200rpm centrifugal condition substratum 5 minutes to remove cell debris.After dull and stereotyped washing and the conditioned culture media preparation, IL-6 standard substance (concentration is 1000pg/ml-1pg/ml) and 170 μ l conditioned culture medias are added the ELISA flat board, incubation is 90 minutes under the room temperature.
Behind the incubation, take out as previously mentioned sample and washing flat board.The 200ng/ml IL-6 of reagent thinner preparation is detected Ab (100 μ l/ hole) adding flat board, and incubation is 60 minutes under the room temperature.Then washing is dull and stereotyped as previously mentioned.Add and use
Test damping fluid (Pa Jin Elmer Co., Ltd) is made 100 μ l Streptavidin europiums of dilution in 1: 1000, and incubation is 60 minutes under the room temperature.Then use
Dull and stereotyped 7 times of lavation buffer solution washing ELISA adds 100 μ l
Toughener (Pa Jin Elmer Co., Ltd).According to manufacturer's working instructions, utilize the fluorescence of dull and stereotyped reader (Pa Jin Elmer Co., Ltd) the quantitative assay 615nm of Victor.
Produce the typical curve of each dull and stereotyped IL-6 standard substance, calculate the IL-6 content that exists in each hole with this, with Excel (Microsoft) analytical data.Not having the inhibitor contrast and do not have also to utilize the IL-6 value in the presence of the IL-6 contrast, discharge Percentage Criterion inhibitor data according to the highest IL-6.Carry out other analysis with Prism (figure pad company), wherein the data mapping is % control reaction and log (scFv/IgG concentration).Utilize Prism curve fitting software (figure pad company) to measure IC
50Value.
Detect the HT1080 test cell line that collaborative IL-6 discharges
The HT1080 IL-17 that this test is described before being induces the improved form of IL-6 release test, and wherein IL-6 reacts and collaborative the generation to human il-17 A and the human TNF alpha (RD system house) that adds.
With the titrating solution of phosphate-buffered saline (PBS) preparation purifying scFv/IgG, 37 ℃ in test medium with IL-17A (125pM final concentration) preincubation 30-60 minute.Substratum is detached cell, add simultaneously the 100 μ l/ hole test mediums that contain human TNF alpha (TNF α final concentration is 25pM).To wherein adding 100 μ l/ hole samples, (18 hours ± 4 hours) are incubated overnight.Collect supernatant liquor, check immediately IL-6 or be stored in-20 ℃.
Detect the IL-6 level by ELISA, HT1080 IL-17 induces the described analytical data of IL-6 release test as mentioned.
Producing natural IL-17A and antibody induces the HT1080 cell to produce to T cell conditioned culture media
Effect
From 3 blood donors the 200m1 human blood is collected in the blood bag that contains heparin.Blood is layered on the LSM (in Greiner Leucosep test tube, 30ml blood is layered on the 15ml LSM) centrifugal (2000rpm, 20 minutes, room temperature, brakeless).Get blood plasma and add fresh test tube, aseptic 0.9% salt solution of centrifugal usefulness is prepared into 5% autologous plasma.Cell (PBMC) layer of getting the monokaryon on surface adds 4 50ml centrifuge tubes, with substratum (having added the RPMI of 10%hiFCS, glutamine (2mM), penicillin (100U/ml) and Streptomycin sulphate (0.1mg/ml) and 1% non-essential amino acid) volume in each test tube is doubled.
Centrifugal PBMC (1800rpm, 10 minutes, room temperature) is resuspended in each cell precipitation thing in about 15ml 5% blood plasma, merges by each blood donor., then be resuspended in the 2ml substratum to reduce number of platelets with 5% blood plasma washing PBMC three times (1000rpm, 10 minutes).
PBMC is added nylon wool post (polymerization scientific company (Polysciences) #425A, be immersed in first in the substratum and be warmed to 37 ℃ re-use), incubation (1 hour, 37 ℃).Then in post, drip the 20ml substratum, collect the effluent liquid that contains purifying T cell.
There is being anti--CD3/ to resist-CD28 (Sigma company (Sigma), final concentration is 1 μ g/ml), TGF β (RD system house, 5ng/ml), IL-23 (RD system house, 50ng/ml), anti--IFN γ (RD system house, 10 μ g/ml) and goat anti--mouse IgG linking agent (Sigma company, 4 μ g/ml) existence is lower, and stimulating final concentration is 5 * 10
6The T cell of cell/ml.Add anti--CD3/ anti--left standstill first 15 minutes behind the CD28, add again linking agent.37 ℃ stimulated the T cell 48 hours.Centrifugal (1000rpm, 5 minutes) cell is removed supernatant liquor.
In substratum (having added the DMEM of 10%hiFCS, glutamine (2mM), penicillin (100U/ml) and Streptomycin sulphate (0.1mg/ml) and 1% non-essential amino acid), with the HT1080 cell with 1.5 * 10
4Cells/well is inoculated in 96 orifice plates (Rodrigo Costa company), with 37 ℃ and 5%CO
2Moistening atmosphere overnight incubation.
The titrating solution of Dispersal risk in substratum mixes isopyknic antibody-solutions with conditioning T cell culture medium as mentioned above, incubation (37 ℃, 1 hour).Isolation medium and cell are also replaced with antibody-solutions, and cell is incubated overnight.Collect supernatant liquor ,-20 ℃ preserve until as described (Cytoset, biogenic company) use the IL-6 elisa assay.With the reacting phase ratio that does not have under the antibody existence, the Origin v7.5 of employing origin laboratory company (OriginLab Corporation) maps to the IL-6 value and calculates the IC that suppresses IL-6 release
50Value.
Chondrocyte IL-6/IL-8/MMP13/PGE
2
-release test
Under the approval of local Ethics Committee, behind the knee prosthesis of Human Osteoarthritis, obtain cartilage.37 ℃, spend the night with 2mg/ml collagenase digesting sample.After the digestion, the chondrocyte that increases in the culturing bottle that contains the improved Eagle substratum of Dulbecco has added 10% foetal calf serum, penicillin (100 unit), Streptomycin sulphate (0.1mg/ml), L-glutaminate (2mM), amphotericin B (2.5 μ g/ml) and non-essential amino acid (1X) in the described substratum.Cultivate 4 independently clones, use the first-generation.Collect the chondrocyte, in substratum with 1.5 * 10
4The density of cells/well is inoculated into 96 orifice plates, 37 ℃, 5%CO
2Under leave standstill to adhere to and spend the night.
With test medium (having added 2% foetal calf serum, penicillin (100 unit), Streptomycin sulphate (0.1mg/ml), L-glutaminate (2mM) and the improved Eagle substratum of the Dulbecco of non-essential amino acid (1X)) will resist-the IL-17A antibody dilution becomes various concentration (0.08-400nM does not wait).With test medium dilution recombinant human IL-17A (inside or Pai Puluo company intestinal bacteria derive), with final concentration 0.2 and 2nM test.The IL-17A that will recombinate adds anti--IL-17 antibody-solutions, preincubation 1 hour.With substratum and chondrocyte isolation, add 100 μ l restructuring IL-17/ and resist-the IL-17A mixtures of antibodies.Incubation was collected culture supernatant after 24 hours.Detect people IL-6, IL-8, MMP13 and PGE by ELISA
2
Detect the ELISA that people IL-6 produces:
According to manufacturer's working instructions, by ELISA, (IL-6 CHC1264) detects to Cytoset in biogenic company.In brief, 4 ℃ are spent the night with the coated ELISA flat board of mono-clonal IL-6 antibody (1 μ g/ml), with the sealing of 1%BSA/ bicarbonate buffer.Wash with 0.05% polysorbas20/PBS dull and stereotyped, under the room temperature with the culture supernatant incubation of the little mouse-anti of human chondrocytes and vitamin H-coupling-huIL-6 antibody (0.4 μ g/ml) 2 hours.After the washing, utilize the Streptavidin (1: 2500) of horseradish peroxidase to detect IL-6.Utilize the TMB colour developing dull and stereotyped.Use 2M H
2SO
4Termination reaction utilizes the dull and stereotyped reader of PHERAstar to measure the 450nm optical density(OD).
Detect the ELISA that people IL-8 produces:
According to manufacturer's working instructions, by ELISA, (IL-8 CHC1304) detects to Cytoset in biogenic company.In brief, 4 ℃ are spent the night with the coated ELISA flat board of mono-clonal IL-8 antibody (1 μ g/ml), with the sealing of 1%BSA/ bicarbonate buffer.Wash with 0.05% polysorbas20/PBS dull and stereotyped, under the room temperature with the culture supernatant incubation of the little mouse-anti of human chondrocytes and vitamin H-coupling-huIL-8 antibody (0.1 μ g/ml) 2 hours.After the washing, utilize the Streptavidin (1: 5000) of horseradish peroxidase to detect IL-8.Utilize the TMB colour developing dull and stereotyped.Use 2M H
2SO
4Termination reaction utilizes the dull and stereotyped reader of PHERAstar to measure the 450nm optical density(OD).
Detect the ELISA that people MMP13 produces:
4 ℃, in phosphoric acid buffer, spend the night with the coated ELISA flat board of little mouse-anti-huMMP13 monoclonal antibody (RD system house, MAB511:1 μ g/ml).Dull and stereotyped with the sealing of 2%BSA/ phosphoric acid buffer, then use 0.05% polysorbas20/PBS washing, 37 ℃ add rabbit with human chondrocytes and resist-huMMP13 polyclonal antibody (Abcam 9128:1: culture supernatant incubation 2500 diluents) 2 hours.After the washing, utilize resisting-family's rabbit igg (peace agate West Asia NA934:1 of Biological Science Co., Ltd: 2500) detect MMP13 of horseradish peroxidase.Utilize the TMB colour developing dull and stereotyped.Use 2M H
2SO
4Termination reaction utilizes the dull and stereotyped reader of PHERAstar to measure the 450nm optical density(OD).
Detect people PGE
2The ELISA that produces:
4 ℃, spend the night with the coated ELISA flat board of little mouse-anti-family's rabbit igg (Sigma company, R2004:10 μ g/ml).Wash with 0.05% polysorbas20/PBS dull and stereotyped, under the room temperature with human chondrocytes, prostaglandin A chE tracer agent (Kaman's chemical company (Cayman Chemicals), 414010) and PGE
2The culture supernatant of antiserum(antisera) (inner preparation, 1: 50,000) is incubated overnight.After the washing, utilize ellman's reagent (Kaman's chemical company, 400050) colour developing dull and stereotyped, utilize the dull and stereotyped reader of PHERAstar to measure the 450nm optical density(OD).
The IL-17 Synergism Testing in the cartilage explant after death
Under complete ethics approval, obtain to maintain after death knee joint sample in the improved Eagle substratum of Dulbecco (DMEM) from the Nottingham through Mil hospital (Kings Mill Hospital, Nottingham).Under aseptic condition, dissect each knee joint with No. 22 scalpels.May part dark sheet (full depth pieces) be being helped in the cartilage separation.Utilize cork cork drill (5mm) cutting explant sheet, in 220 μ l substratum (having added the DMEM (without phenol red) of penicillin (100U/ml), Streptomycin sulphate (0.1mg/ml), glutamine (2mM), 1% non-essential amino acid and gentamicin (1 μ g/ml)), inoculate 96 orifice plates according to 1 explant/hole.Explant leaves standstill first to stimulate in 3 days again.Each condition has the explant of 12 repetitions.
Used stimulation is the combination of the TNF α (Pai Puluo technology company) of the IL-17A (Pai Puluo technology company) of alone substratum, alone 10ng/ml, alone 1ng/ml or the dilution of the substratum of all using sulfur acid PXB (2 μ g/ml).For initial experiment, will resist IL-17A neutralizing antibody and their isotype contrast (mouse IgG with the substratum that contains 2 μ g/ml Polymyxin B-sulfate USPs
1(RD system house), human IgG
1CAT002 (Cambridge Antibody Tech)) be diluted to 50nM, and for IC
50Experiment is diluted to 0.5-50nM.With antibody with shown in cytokine mix, all solution elder generation incubations add explant (37 ℃, 30 minutes) again.
Isolation medium and explant stimulate substratum to replace with 220 μ l.Dull and stereotyped 72 hours (37 ℃, in the moistening atmosphere) of elder generation's incubation take out supernatant liquor again, are stored in-20 ℃.Working instructions and embodiment 3 (avidity of antibody on human IL-17A) according to the manufacturer are described, analyze IL-6 content by ELISA (Cytoset, biogenic company).Adopt Origin v7.5 (origin laboratory company) evaluation.
In the RA synovioblast, suppress IL-17A/TNF α and induce IL-8
Under ethics was ratified fully, the rheumatoid arthritis knee sample of total joint replacement was at AstraZeneca R ﹠amp; D Charnwood obtains.Give AstraZeneca R ﹠amp with 2nd generation RA synovioblast; D Alderley Park.With cell at T225cm
2Cultivate in the flask, be equipped with in the flask that to have added 10%FCS (extra large cloning companies (Hyclone)), penicillin (100U/ml), Streptomycin sulphate (0.1mg/ml), glutamine (2mM), 1% non-essential amino acid and gentamicin (1 μ g/ml) be DMEM, in the humidification incubator, maintain 37 ℃.
Per two all passage cells once used for the 5th generation.Utilize trypsinase-EDTA that cell and flask are broken away from, with 5 * 10
3Cells/well is inoculated in 96 orifice plates, inoculates 3 days and uses afterwards.
Used stimulation be alone substratum, alone IL-17A (Pai Puluo technology company) or with the combination of TNF α (RD system house).To resist IL-17A neutralizing antibody and their isotype contrast (mouse IgG with the substratum that contains 1 μ g/ml PXB
1(RD system house), human IgG
1CAT002 (Cambridge Antibody Tech)) is diluted to 50nM.With antibody with shown in cytokine mix, all solution elder generation incubations add inoblast (37 ℃, 30 minutes) again.
Isolation medium and cell stimulate substratum to replace with 100 μ l.Dull and stereotyped 24 hours (37 ℃, in the moistening atmosphere) of elder generation's incubation take out supernatant liquor again, are stored in-20 ℃.Working instructions and embodiment 3 (avidity of antibody on human IL-17A) according to the manufacturer are described, analyze IL-8 content by ELISA (Cytoset, biogenic company).Adopt Origin v7.5 (origin laboratory company) that IL-8 numerical value is mapped to obtain IC
50Value.
PA
2
Analyze
The main pharmacy instrument of the avidity of quantitative assay competitive antagonist is that Schild analyzes.Adopt this scheme, the system that can determine in the function test assessment antagonist avidity is the dependency mode not.The following concept of the method foundation: antagonist concentration and avidity thereof determine the antagonistic action of agonist reaction.Because it is known to quantize the concentration of antagonistic action and antagonist, can measure the avidity of antagonist.By have and do not have in the presence of the antagonist to detect the agonist isoreactivity concentration that is called dose ratio (DR) recently quantize this antagonistic action.
There is not agonist (normally IL-17A) EC in the presence of the binding members in utilization
50EC with lower this agonist of binding members existence that single concentration is arranged
50Than calculating dose ratio.Then can will be expressed as the dose ratio of log (DR-1) be used for the log[binding members] linear regression, return thereby produce Schild.Therefore, every kind of binding members concentration has corresponding DR value; It is that log (DR-1) is to the log[binding members that these DR values are mapped] return.According to the Schild equation, if antagonistic action is emulative, then at log (DR-1) and log[binding members] between linear relationship should be arranged, this equation is as follows:
Log(DR-1)＝log[B]-log K
B
[B] is the volumetric molar concentration of binding members.
Under these situations, coordinate 0 value can obtain log[B]=log K
BThe time x-axle intercept.Therefore, the binding members concentration of generation log (DR-1)=0 equals log K
B, the equilibrium dissociation constant of binding members-receptor complex.This system does not rely on the estimated value that quantizes binding members avidity.This scheme is generally used for measuring the avidity of receptor antagonist, however according to the similar hypothesis of part neutralizing effect, the calculated value of dose ratio also should be able to estimate in the binding members and cell on the avidity of IL-17 activity.Because obtain K from logarithmic graph
BValue, they normally logarithm distribute.The negative logarithm of this certain concentration is called pA empirically
2, antagonist makes the agonist dose response curve produce the concentration of twice displacement.Can be by calculating the pA of single concentration antagonist
2, produce the single value of dose ratio from following equation, render a service thereby can quantize antagonist:
pA
2＝log(DR-1)-log[B]
[B]=cause initial inferior highest response and antagonist volumetric molar concentration that agonist concentration is doubled.
DR=quantizes dose ratio by the ratio in the agonist isoreactivity concentration that has and do not have to detect in the presence of the antagonist.
Can calculate pA from the dose-response testing data
2
Be used for pA
2Analysis is with the test method of calculating antibody to the avidity of human il-17 A
(European cell cultures preservation institute is ECACC) with 5 * 10 with the HT1080 cell
4Cells/well is inoculated in the flat tissue culture test panel in 96-hole (Rodrigo Costa company).Then with cell in 37 ℃ and 5%CO
2Humidification atmosphere in, in 200 μ l test medium/holes (MEM that contains hot deactivation Australia's FBS, 1% (v/v) the MEM-non-essential amino acid of Earles salt and L-glutaminate, 10% (v/v) (without L-glutaminate), hero company)) middle overnight incubation.
With the titrating solution (final concentration is 1 μ M-7pM) of phosphate-buffered saline (PBS) preparation IL-17A, 37 ℃ in test medium with the IgG (used Ab final concentration is 270nM-270pM) of purifying preincubation 30-60 minute.Substratum is detached cell, add simultaneously 100 μ l/ hole test mediums.To wherein adding 100 μ l/ hole samples, (18 hours ± 4 hours) are incubated overnight.Collect supernatant liquor, check immediately or be stored in-20 ℃.
Utilize the IL-6 level in Hu IL-6 Duoset ELISA test kit (RD system house) the mensuration supernatant liquor.On with the coated FluoroNunc Maxisorb flat board that spends the night of 2 μ g/ml IL-6 capture antibodies (Ab) (with PBS dilution, 100 μ l/ holes), carry out ELISA.Wash ELISA dull and stereotyped three times with the PBS-tween, add 300 μ l reagent thinners (1% bovine serum albumin (BSA) of PBS preparation).The lower incubation of room temperature (RT) washs the ELISA flat board after 1 hour as previously mentioned.Between 1 hour incubation period, with 1200rpm centrifugal condition substratum 5 minutes to remove cell debris.After dull and stereotyped washing and the conditioned culture media preparation, IL-6 standard substance (concentration is 1000pg/ml-1pg/ml) and 170 μ l conditioned culture medias are added the ELISA flat board, incubation is 90 minutes under the room temperature.Behind the incubation, take out as previously mentioned sample and washing flat board.The 200ng/ml IL-6 of reagent thinner preparation is detected Ab (100ml/ hole) adding flat board, and incubation is 60 minutes under the room temperature.Then washing is dull and stereotyped as previously mentioned, adds to use
Test damping fluid (Pa Jin Elmer Co., Ltd) is made 100 μ l Streptavidin europiums of dilution in 1: 1000, and incubation is 60 minutes under the room temperature.Then wash ELISA dull and stereotyped 7 times with the DELFIA lavation buffer solution, add 100 μ l DELFIA tougheners (Pa Jin Elmer Co., Ltd).According to manufacturer's working instructions, utilize the fluorescence of dull and stereotyped reader (Pa Jin Elmer Co., Ltd) the quantitative assay 615nm of Victor.
Produce the typical curve that adds each dull and stereotyped IL-6 standard substance, calculate the IL-6 content that exists in each hole with this, with Excel (Microsoft) analytical data.Not having to utilize the IL-6 value in the presence of the inhibitor contrast, discharge Percentage Criterion concentration dependent curve according to the highest IL-6.Carry out other analysis with Prism (figure pad software company), wherein the data mapping is % control reaction and log (IgG concentration), utilizes Prism curve fitting software (figure pad company) to produce IC
50Value.The Hillslope of concentration dependent curve averages and determines with the calculating dose ratio.With these generation Schild figure (still utilizing Prism) and from pA
2Value estimation Kd value.
Binding members disturbs the H/D exchange velocity of IL-17A
Used mark IL-17A 5.0mM Tris.HCl pH7.4,150mM NaCl is mixed with 0.5mg/ml.Used antibody is mixed with 10.6mg/ml with PBS (1.54mM KH2PO4,2.71mM Na2HPO4,155mMNaCl pH7.2).According to manufacturer's working instructions, with antibody coupling in POROS AL resin (Applied Biosystems, Inc.) to prepare 100 μ l posts, be maintained at 1 ℃.
With the 50mM Tris.HCl pH7.5 of 75%D2O preparation, 150mM NaCl washing column.Damping fluid with 75%D2O preparation is diluted to 0.125mg/ml with IL-17A, and incubation 150,500,1500 and 15000 seconds is expelled on the antibody column again.0.2ml damping fluid with the H2O preparation is washed post fast, then the identical time of incubation.Namely 150 seconds sample of exchange exchanges 150 seconds in H2O in D2O, and 500,1500 and 5000 seconds sample is also similar.By injecting the one 80 μ l, then inject 40 μ l, 0.8% formic acid at the different time points wash-out.One 40 μ l after collecting, 1 ℃ adds 20 μ l 2M ureas, 1M TCEP pH3.Whole mixture is expelled on the 100 μ l posts, this post contains stomach en-so that protein digestion is become peptide, thereby can utilize the acetonitrile gradient of 12-28.5% to separate by rpHPLC and utilize Thermo FinniganLCQ electrospray mass spectrometer and Micromass Q-TOF mass spectrograph quality measurement.Adopt the sequence of the SEQUEST software program evaluation parental generation peptide ion of San Jose TF company (Thermo Finnigan, San Jose, CA).
Except following exception, basically measure as mentioned above antibody to the impact of the exchange velocity of IL-17 different piece: at first with containing H
2The O damping fluid is diluted to 0.125mg/ml with IL-17, is then injected into to use H
2The 50mM Tris.HCl pH7.5 of O preparation is on the post that 150mM NaCl washs in advance.In conjunction with after, use H
2The 50mM Tris.HCl pH7.5 of O preparation, 150mM NaCl washes post, then with 75%D
2The 50mM Tris.HCl pH7.5 of O preparation, 150mM NaCl incubation 150,500,1500 and 5000 seconds exchanges back H again
2O also processes as mentioned above.
The clone of mutant IL-17A construction, expression and purifying
Adopt
Technology (hero company) and conventional mutafacient system (utilizing Stratagene multidigit point mutagenesis kit) or polymerase chain reaction clone contain the terminal histidine-tagged mutant IL-17A of C-.Adopt LR Clonase reaction with clone's gene insertion expression vector pCEP4.
At HEK293/EBNA cells wild-type and the mutant IL-17A that contains the terminal His label of C-.Then when cell reaches 60-70% and converges with 30 μ g DNA/T162 flask transfectional cells.Utilize PEI transfection reagent (Sigma company), cell is incubated overnight and removes substratum again, replaces to the fresh culture that does not contain serum, and incubation is 72 hours again.Collect the supernatant liquor of these cells, be used for each recombinant protein of purifying.
Collect the supernatant liquor of the HEK293/EBNA cell of transfection, pH regulator to 7.4 is flowed (GE keep healthy company) soon with the Ni-agarose again pass through affinitive layer purification.With the albumen of PBS damping fluid dialysis purifying ,-Histidine antibody anti-by utilizing is analyzed as SDS-PAGE, Edman N-end sequencing and the western blotting of testing tool.Confirm first the homogeny of protein, begin again combination and functional study.Measure concentration in conjunction with the Coomassie blue stain intensity on 280nm absorbancy and the SDS-PAGE.
The BIAcore of wild-type and mutant IL-17A is in conjunction with experiment
According to manufacturer's working instructions, utilize BIAcore 3000 and amine-coupling reagent kit (BIAcore catalog number (Cat.No.) BR-1000-50) antibody to be fixed in the surface (BIAcore catalog number (Cat.No.) BR-1033-99) of CM5 sensor chip with 100 μ g/ml.Utilize 10mM glycine pH 1.5, produce baseline by acid regeneration.Before each experiment, set up first baseline, the speed of part (with 25nM) with 10 μ l/ minutes was expelled on the surface of chip during 7 minutes.By detecting the rear 60 seconds signal (RU) of front 30 seconds of injection and injection and from the latter, deducting the IL-17A amount that the former measures combination.
HT1080 IL-17 induces IL-6 to discharge (epitope mapping research)
With HT1080 cell (European cell cultures preservation institute, ECACC-ECACC number 85111505) with 5 * 10
4Cells/well is inoculated in the flat tissue culture test panel in 96-hole (Rodrigo Costa company).Then with cell in 37 ℃ and 5%CO
2Humidification atmosphere in, overnight incubation in 200 μ l test mediums (contain L-glutaminate (hero company), 1% penicillin and Streptomycin sulphate, 10% (v/v) heat-inactivated fetal bovine serum (FBS) (extra large cloning companies), 1% (v/v) do not contain the improved Eagle substratum of Dulbecco (DMEM) of the MEM-non-essential amino acid (hero company) of L-glutaminate).
Prepare the titrating solution (final concentration is 50-0.03nM) of interested antibody or contrast IgG with test medium, 37 ℃ with the mutant form of IL-17A or IL-17A (final concentration is as showing shown in the 20b 1-2nM) preincubation 30-60 minute.Prepare other titration curve of independent IL-17A or mutant IL-17A with the reactivity of assessment cell to part.Substratum is detached cell, add cultivation antibody and the IL-17A (or mutant IL-17A) in 100 μ l/ holes, or independent IL-17A, (18 hours ± 4 hours) are incubated overnight.Collect supernatant liquor, check immediately or be stored in-20 ℃.Data point in the experiment is triplicate.
Such as " chondrocyte IL-6/IL-8/MMP13/PGE
2Release test " IL-6 level in the described mensuration supernatant liquor of materials and methods.
Adopt " standard verification and the calculating of unknown material " from the typical curve unknown numerical value of extrapolating, adopt " dose response-1-15 group " among the origin laboratory Origin v7.5 of company to calculate IC
50
Be used for generation and the analysis of the antibody 7Fab of X-radiocrystallography
The antibody 7 of digestion purifying as follows is to produce the Fab fragment:
-preparation is dissolved in the digestion damping fluid of the 30mM DL-cysteine hydrochloride of GIBCO PBS (hero company).
-obtain 10mg/mL solution with the stomach en-(Sigma company) of digestion damping fluid reconstruction papaya latex, be maintained at room temperature and re-used in 30 minutes.
-halfcystine is added IgG to obtain 30mM solution.
-stomach en-is added IgG with the 1mg stomach en-than 100mg IgG.
Add 0.5M iodo-acid amide (Sigma company) after-4 hours and stop digestion, thereby in final digestion mixture, obtain the 50mM iodo-acid amide.
Then purifying Fab becomes PBS pH 7.2 with buffer-exchanged, is concentrated into about 10mg/ml, is used for following X-radiocrystallography.
The X-ray crystal structure is measured: protein preparation, crystallization and crystallography
At expression in escherichia coli recombinant human IL-17A, adopt method (Rudolph etc. the 1996) isolation of occlusion bodies of knowing.Under the room temperature the dissolving damping fluid in homogenate dissolved inclusion body protein (50mMTris pH 8.5,6M guanidine HCl, 10mM DTT) in 1 hour.At room temperature be added dropwise to refolding damping fluid (0.1M CAPSO pH 9.5,0.9M arginine and 0.3: 0.03mM reduction: the gsh of oxidation) make rapid dilution and vigorous stirring, hold over night, thereby with the protein of 0.5mg/ml final concentration refolding dissolving.Utilize 10KDa MWCO spiral-tube (spiral cartridge) (Amicon proflux
TMM12 cuts streaming system) with 5 times of the protein compressions of refolding.Utilize the concentrated protein of S75 (GE keep healthy Biological Science Co., Ltd) size exclusion column purification of size exclusion damping fluid (50mM TrispH 7.4,0.15M NaCl) balance.
The Ettan LC (GE keep healthy company) that utilization is equipped with Superdex 200 PC 3.2/30 post (GE keep healthy company) analyzes gel-filtration causes IL-17A/Fab mixture productive rate maximum with evaluation ratio of mixture.Use first gel-filtration damping fluid (BTP 25mM, pH 7.0, NaCl 100mM) balance columns, loaded 35 μ l samples in 1/ minute with 50 μ again.Utilize described material, the IL-17A of 3.6: 1 ratios: Fab produces the maximum composite productive rate.Then with this ratio IL-17A and Fab are mixed, concentrated with 500 μ l 30kDa molecular weight cutoff value centrifugal concentrating devices (VS company (Viva Spin)) of 1ml gel-filtration damping fluid washing before utilizing.Protein compression is reduced to OD280=7.6, then takes out 50 μ l samples.Further concentrated remaining until OD280=16.6.20 ℃, utilize the drop of Nextal pallet and two kinds of protein concns of 2 μ l+2 μ l to carry out hanging drop vapor diffusion crystallization (hanging drop vapourdiffusion crystallisation).
2-3 is after week, diffraction quality crystal (diffraction quality crystal) is from containing 90.9mM PCTP pH of buffer 4.0,9.1mM PCTP pH of buffer 9.5 (1), 100mM ammonium sulfate is grown in the high density hanging drop of 14%w/vPEG3350.Collect crystal, with (-) (-) butane 2,3 glycol (with the 20%v/v of respective aperture solution preparation) cryoprotection, utilize Oxford Cryostream with the 100K quick freezing.The diffraction quality of the Rigaku Fre rotation anion generator screening crystal of Saturn 944 CCD detectors and the freezing head of Xstream (cryo head) is equipped with in utilization.Preserve rapidly those crystal that show orderly diffraction, utilize ESRF, collect data at ID-29.
Utilize European synchrotron light equipment (ESFR) to be collected in the diffraction data of the single crystal of 100K cooling, baseline is ID29, utilizes ADSC Quantum Q315r detector.Be recorded in respectively in the rotating frame of 0.75 ° and 0.2 ° that wavelength is 0.9787 on the angular range of 225 ° and 155 °
Two data sets.Adopt the view data of MOSFLM (Leslie, 1991) each data set of integration, utilize the program of CCP4 external member (CCP4,1994) to merge separately and convergent-divergent.Get rid of inner R in the final convergent-divergent and merge＞20%/batch image.Data gathering statistics sees Table 23.
All crystals belongs to oblique crystal spacer P2
1, have typical cell size a=98.5
B=66.7
And c=203.8
β=91.7 ° wherein.The crystallography asymmetry unit contains 4 Fab molecules and 2 IL-17A molecules, is 2.68 thereby cause the Matthews coefficient corresponding to solvent 54%
3/Da (Matthews, 1968).Utilize CCP4 program MOLREP (CCP4,1994), measure the IL-17A/Fab composite structure by the molecular replacement method.In the first test, can locate the also variable domain (the residue 2-110 of light chain, the residue 2-124 of heavy chain) of the Fab of directed 3 molecules (obtaining Faber etc., 1998 from PDB clauses and subclauses 1AQK).Subsequently, determine the position of these Fab variable domains, the constant domain (the residue 111-216 of light chain, the residue 125-226 of heavy chain) of location and directed 3 molecule Fab.The correct Fab molecule that produces shows that molecular replacement solution is correct, and can with the 4th and last Fab molecule place the asymmetry unit of lattice.Adopt REFMAC (Murshudov etc., 1997), utilize the isotropy Factor B to optimize limited maximum likelihood and obtain Initial R
Work/ R
FreeBe 33.6%/41.5%.Adopt Molecular Graphs program COOT (Emsley and Cowtan, 2004) the manual Fab of reconstruction model and the correct aminoacid sequence of displacement.Identify the IL-17A molecule by the FoFc electron density map that visual inspection is residual.Place the 2FoFc electron density that does not occupy by hand by the chain with all lengths, can identify the conservative amino acid motif [50-RSTSPW-57] of IL-17A, and can beyond all doubt simulation.So can the superpose model (PDB clauses and subclauses 1JPY, Hymowitz etc., 2001) of IL-17F, thus can the rapid interpretive dimer molecule.This stack has also been made clear in the molecule and has been interacted and orderly part with Fab, and the opposite end of the dimer molecule that prolongs is not seen in electron density, and namely it is unordered.The IL-17A model can make up electron density as much as possible.Adopt REFMAC with R
Work/ R
FreeBe 27.2%/33.4% optimize limited maximum likelihood one and take turns after, correct aminoacid sequence manual rebuild and arrangement is applied to the IL-17A homodimer.After last that adopts MOLREP taken turns MR, the IL-17A homodimer of reconstruction (the visible residue is the 39-129 of A chain and the 44-132 of B chain in the electron density) was used for directed and location the 2nd IL-17A homodimer.Adopt first REFMAC to optimize IL-17A Fab composite structure (containing 4 Fab molecules and 2 IL-17A molecules) and make it to assemble, adopt again manual water molecules and the sulfate ion of adding of COOT.Final the R-factor, R
WorkAnd R
FreeRespectively 22.7% and 28.4%.
Table 23. crystal parameter and X-ray data-processing and optimization statistics
R
FreeIt is the cross validation R factor of 5% unique reflection measurement group of calculating
Sequence
The VH structural domain of binding members, VL structural domain and CDR sequence are shown in the sequence table of enclosing, and wherein SEQ ID NO is corresponding to following:
The PTN=aminoacid sequence
1 antibody, 01 VH DNA
2 antibody, 01 VH PTN
3 antibody, 01 VH CDR1 PTN
4 antibody, 01 VH CDR2 PTN
5 antibody, 01 VH CDR3 PTN
6 antibody, 01 VL DNA
7 antibody, 01 VL PTN
8 antibody, 01 VL CDR1 PTN
9 antibody, 01 VL CDR2 PTN
10 antibody, 01 VL CDR3 PTN
11 antibody, 02 VH DNA
12 antibody, 02 VH PTN
13 antibody, 02 VH CDR1 PTN
14 antibody, 02 VH CDR2 PTN
15 antibody, 02 VH CDR3 PTN
16 antibody, 02 VL DNA
17 antibody, 02 VL PTN
18 antibody, 02 VL CDR1 PTN
19 antibody, 02 VL CDR2 PTN
20 antibody, 02 VL CDR3 PTN
21 antibody, 03 VH DNA
22 antibody, 03 VH PTN
23 antibody, 03 VH CDR1 PTN
24 antibody, 03 VH CDR2 PTN
25 antibody, 03 VH CDR3 PTN
26 antibody, 03 VL DNA
27 antibody, 03 VL PTN
28 antibody, 03 VL CDR1 PTN
29 antibody, 03 VL CDR2 PTN
30 antibody, 03 VL CDR3 PTN
31 antibody, 04 VH DNA
32 antibody, 04 VH PTN
33 antibody, 04 VH CDR1 PTN
34 antibody, 04 VH CDR2 PTN
35 antibody, 04 VH CDR3 PTN
36 antibody, 04 VL DNA
37 antibody, 04 VL PTN
38 antibody, 04 VL CDR1 PTN
39 antibody, 04 VL CDR2 PTN
40 antibody, 04 VL CDR3 PTN
41 antibody, 05 VH DNA
42 antibody, 05 VH PTN
43 antibody, 05 VH CDR1 PTN
44 antibody, 05 VH CDR2 PTN
45 antibody, 05 VH CDR3 PTN
46 antibody, 05 VL DNA
47 antibody, 05 VL PTN
48 antibody, 05 VL CDR1 PTN
49 antibody, 05 VL CDR2 PTN
50 antibody, 05 VL CDR3 PTN
51 antibody, 06 VH DNA
52 antibody, 06 VH PTN
53 antibody, 06 VH CDR1 PTN
54 antibody, 06 VH CDR2 PTN
55 antibody, 06 VH CDR3 PTN
56 antibody, 06 VL DNA
57 antibody, 06 VL PTN
58 antibody, 06 VL CDR1 PTN
59 antibody, 06 VL CDR2 PTN
60 antibody, 06 VL CDR3 PTN
61 antibody, 07 VH DNA
62 antibody, 07 VH PTN
63 antibody, 07 VH CDR1 PTN
64 antibody, 07 VH CDR2 PTN
65 antibody, 07 VH CDR3 PTN
66 antibody, 07 VL DNA
67 antibody, 07 VL PTN
68 antibody, 07 VL CDR1 PTN
69 antibody, 07 VL CDR2 PTN
70 antibody, 07 VL CDR3 PTN
71 antibody, 08 VH DNA
72 antibody, 08 VH PTN
73 antibody, 08 VH CDR1 PTN
74 antibody, 08 VH CDR2 PTN
75 antibody, 08 VH CDR3 PTN
76 antibody, 08 VL DNA
77 antibody, 08 VL PTN
78 antibody, 08 VL CDR1 PTN
79 antibody, 08 VL CDR2 PTN
80 antibody, 08 VL CDR3 PTN
81 antibody, 09 VH DNA
82 antibody, 09 VH PTN
83 antibody, 09 VH CDR1 PTN
84 antibody, 09 VH CDR2 PTN
85 antibody, 09 VH CDR3 PTN
86 antibody, 09 VL DNA
87 antibody, 09 VL PTN
88 antibody, 09 VL CDR1 PTN
89 antibody, 09 VL CDR2 PTN
90 antibody, 09 VL CDR3 PTN
91 antibody, 10 VH DNA
92 antibody, 10 VH PTN
93 antibody, 10 VH CDR1 PTN
94 antibody, 10 VH CDR2 PTN
95 antibody, 10 VH CDR3 PTN
96 antibody, 10 VL DNA
97 antibody, 10 VL PTN
98 antibody, 10 VL CDR1 PTN
99 antibody, 10 VL CDR2 PTN
100 antibody, 10 VL CDR3 PTN
101 antibody, 11 VH DNA
102 antibody, 11 VH PTN
103 antibody, 11 VH CDR1 PTN
104 antibody, 11 VH CDR2 PTN
105 antibody, 11 VH CDR3 PTN
106 antibody, 11 VL DNA
107 antibody, 11 VL PTN
108 antibody, 11 VL CDR1 PTN
109 antibody, 11 VL CDR2 PTN
110 antibody, 11 VL CDR3 PTN
111 antibody, 12 VH DNA
112 antibody, 12 VH PTN
113 antibody, 12 VH CDR1 PTN
114 antibody, 12 VH CDR2 PTN
115 antibody, 12 VH CDR3 PTN
116 antibody, 12 VL DNA
117 antibody, 12 VL PTN
118 antibody, 12 VL CDR1 PTN
119 antibody, 12 VL CDR2 PTN
120 antibody, 12 VL CDR3 PTN
121 antibody, 13 VH DNA
122 antibody, 13 VH PTN
123 antibody, 13 VH CDR1 PTN
124 antibody, 13 VH CDR2 PTN
125 antibody, 13 VH CDR3 PTN
126 antibody, 13 VL DNA
127 antibody, 13 VL PTN
128 antibody, 13 VL CDR1 PTN
129 antibody, 13 VL CDR2 PTN
130 antibody, 13 VL CDR3 PTN
131 antibody, 14 VH DNA
132 antibody, 14 VH PTN
133 antibody, 14 VH CDR1 PTN
134 antibody, 14 VH CDR2 PTN
135 antibody, 14 VH CDR3 PTN
136 antibody, 14 VL DNA
137 antibody, 14 VL PTN
138 antibody, 14 VL CDR1 PTN
139 antibody, 14 VL CDR2 PTN
140 antibody, 14 VL CDR3 PTN
141 antibody, 15 VH DNA
142 antibody, 15 VH PTN
143 antibody, 15 VH CDR1 PTN
144 antibody, 15 VH CDR2 PTN
145 antibody, 15 VH CDR3 PTN
146 antibody, 15 VL DNA
147 antibody, 15 VL PTN
148 antibody, 15 VL CDR1 PTN
149 antibody, 15 VL CDR2 PTN
150 antibody, 15 VL CDR3 PTN
151 antibody, 16 VH DNA
152 antibody, 16 VH PTN
153 antibody, 16 VH CDR1 PTN
154 antibody, 16 VH CDR2 PTN
155 antibody, 16 VH CDR3 PTN
156 antibody, 16 VL DNA
157 antibody, 16 VL PTN
158 antibody, 16 VL CDR1 PTN
159 antibody, 16 VL CDR2 PTN
160 antibody, 16 VL CDR3 PTN
161 stump-tailed macaque IL-17 DNA
162 stump-tailed macaque IL-17 PTN
163 antibody, 1 VL DNA
164 antibody, 1 VL PTN
165 antibody, 2 VL DNA
166 antibody, 2 VL PTN
167 antibody 3 and 9 VL DNA
168 antibody 3 and 9 VL PTN
169 antibody, 4 VL DNA
170 antibody, 4 VL PTN
171 antibody 5 and 8 VL DNA
172 antibody 5 and 8 VL PTN
173 antibody, 6 VL DNA
174 antibody, 6 VL PTN
175 antibody, 7 VL DNA
176 antibody, 7 VL PTN
177 antibody, 10 VL DNA
178 antibody, 10 VL PTN
179 antibody, 11 VL DNA
180 antibody, 11 VL PTN
181 antibody 12 and 16 VL DNA
182 antibody 12 and 16 VL PTN
183 antibody, 13 VL DNA
184 antibody, 13 VL PTN
185 antibody, 14 VL DNA
186 antibody, 14 VL PTN
187 antibody, 15 VL DNA
188 antibody, 15 VL PTN
189 VH FR1 PTN
190 VH FR2 PTN
191 VH FR3 PTN
192 VH FR4 PTN
193 VL FR1 PTN
194 VL FR2 PTN
195 VL FR3 PTN
196 VL FR4 PTN
The ripe hIL-17 of 197 marks
a
The human il-17 of 198 maturations
a
199 human il-17s
aPeptide
The mutant precursor hIL-17 of 200 marks
a
The human il-17 of 201 mutant maturations
a
Antibody 3,4 and 5 has identical VH structural domain.
Antibody 9,10 and 12 has identical VH structural domain.
Antibody 2 and 10 has identical VL CDR.The VL structural domain sequence of shown antibody 2 is through kind of being, the VL structural domain sequence of shown antibody 10 is not planted being.
Antibody 3,9 and 11 has identical VL structural domain.
Antibody 4 and 15 has identical VL structural domain.
Antibody 5 and 8 has identical VL structural domain.
Antibody 12 and 16 has identical VL structural domain.
The VL structural domain nucleotide sequence of antibody 1-16 is not included in the gag codon that 3 ' end shows among the SEQ ID NO:6,16,26,36,46,56,66,76,86,96,106,116,126,136,146 and 156.Therefore, VL structural domain sequence does not comprise respectively the terminal Glu residue (residue 112) of C-among the SEQ ID NO:7,17,27,37,47,57,67,77,87,97,107,117,127,137,147 and 157.Glu112 residue and corresponding gag codon are not expressed in antibody 1-16.Relatively institute's write sequence is that constant gene segment C shows that Glu residue and corresponding gag codon do not consist of the part of VL structural domain with planting.
111 Gly residue (Kabat residue 108) is present among the scFv and IgG sequence of expression.Yet it is in the j sector sequence that this residue is not present in the ethnic group that forms VL structural domain framework region 4.This Gly residue is not regarded the part of VL structural domain as.
Be to express the light chain of IgG, the nucleotide sequence of encoding antibody light chain is provided, comprise the First Exon of coding VL structural domain, the Second Exon of coding CL structural domain, the intron that First Exon and Second Exon are separated.Under normal conditions, intron is fallen in the montage of cell mRNA processing mechanism, thereby 3 ' end of First Exon is linked to each other with 5 ' end of Second Exon.Therefore, when the DNA with described nucleotide sequence was expressed as RNA, this first and second exons montage together.The RNA that translates this montage can produce the polypeptide that comprises VL and CL structural domain.After the montage, by the Gly of 111 of the first two base (gt) codings of last bases (g) of VL structural domain framework 4 sequences and CL structural domain.
The VL structural domain sequence of antibody 1-16 is shown in above SEQ ID NO:163-188.As last codon ending, Leu is the final amino acid residue of corresponding VL structural domain aminoacid sequence to VL structural domain nucleotide sequence with cat.
Reference
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Sequence table
＜110〉Astrazeneca AB (SE) (AstraZeneca AB)
Cambridge Antibody Tech (Cambridge Antibody Technology Limited)
＜120〉antibody molecule of human il-17
<130>102331
<140>
<141>
<150>US 60/815,828
<151>2006-06-23
<150>US 60/913,566
<151>2007-04-24
<160>201
＜170〉PatentIn version 3 .3
<210>1
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>1
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
atttggggag tggctgggag ctggggccag gggacaatgg tcaccgtctc ctca 354
<210>2
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>2
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Trp Gly Val Ala Gly Ser Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210>3
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>3
Ser Tyr Ala Met Ser
5
<210>4
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>4
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>5
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>5
Asp Leu Ile Trp Gly Val Ala Gly Ser
5
<210>6
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>6
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtctt atgatgacag cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>7
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>7
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp
85 90 95
Ser Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>8
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>8
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>9
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>9
Ala Asn Asn Gln Arg Pro Ser
5
<210>10
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>10
Gln Ser Tyr Asp Asp Ser Ser Val Val
5
<210>11
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>11
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
atttggggag tggctgggag ctggggccag gggacactgg tcaccgtctc ctca 354
<210>12
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>12
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Trp Gly Val Ala Gly Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>13
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>13
Ser Tyr Ala Met Ser
5
<210>14
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>14
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>15
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>15
Asp Leu Ile Trp Gly Val Ala Gly Ser
5
<210>16
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>16
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtt cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240
ctgaagactg aggacgaggc tgactactac tgccagtcgt acgaccccca cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>17
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>17
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Pro
85 90 95
His Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>18
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>18
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>19
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>19
Ala Asn Asn Gln Arg Pro Ser
5
<210>20
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>20
Gln Ser Tyr Asp Pro His Ser Val Val
5
<210>21
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>21
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
attcacgggg tgacgcggaa ctggggccag gggacactgg tcaccgtctc ctca 354
<210>22
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>22
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile His Gly Val Thr Arg Asn Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>23
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>23
Ser Tyr Ala Met Ser
5
<210>24
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>24
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>25
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>25
Asp Leu Ile His Gly Val Thr Arg Asn
5
<210>26
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>26
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcct actcccccca cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>27
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>27
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
His Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>28
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>28
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>29
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>29
Ala Asn Asn Gln Arg Pro Ser
5
<210>30
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>30
Gln Ser Tyr Ser Pro His Ser Val Val
5
<210>31
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>31
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
attcacgggg tgacgcggaa ctggggccag gggacactgg tcaccgtctc ctca 354
<210>32
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>32
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile His Gly Val Thr Arg Asn Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>33
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>33
Ser Tyr Ala Met Ser
5
<210>34
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>34
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>35
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>35
Asp Leu Ile His Gly Val Thr Arg Asn
5
<210>36
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>36
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcct actccccgac gagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>37
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>37
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
Thr Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>38
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>38
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>39
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>39
Ala Asn Asn Gln Arg Pro Ser
5
<210>40
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>40
Gln Ser Tyr Ser Pro Thr Ser Val Val
5
<210>41
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>41
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
attcacgggg tgacgcggaa ctggggccag gggacactgg tcaccgtctc ctca 354
<210>42
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>42
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile His Gly Val Thr Arg Asn Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>43
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>43
Ser Tyr Ala Met Ser
5
<210>44
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>44
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>45
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>45
Asp Leu Ile His Gly Val Thr Arg Asn
5
<210>46
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>46
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtctt ataaccacaa ggacatcgtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>47
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>47
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asn His
85 90 95
Lys Asp Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>48
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>48
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>49
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>49
Ala Asn Asn Gln Arg Pro Ser
5
<210>50
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>50
Gln Ser Tyr Asn His Lys Asp Ile Val
5
<210>51
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>51
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
atttggggag tggctgggag ctggggccag gggacactgg tcaccgtctc ctca 354
<210>52
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>52
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Trp Gly Val Ala Gly Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>53
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>53
Ser Tyr Ala Met Ser
5
<210>54
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>54
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>55
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>55
Asp Leu Ile Trp Gly Val Ala Gly Ser
5
<210>56
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>56
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcgt acagcccgag cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>57
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>57
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
Ser Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>58
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>58
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>59
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>59
Ala Asn Asn Gln Arg Pro Ser
5
<210>60
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>60
Gln Ser Tyr Ser Pro Ser Ser Val Val
5
<210>61
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>61
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
attcacgggg tgacgcggaa ctggggccag gggacactgg tcaccgtctc ctca 354
<210>62
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>62
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile His Gly Val Thr Arg Asn Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>63
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>63
Ser Tyr Ala Met Ser
5
<210>64
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>64
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>65
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>65
Asp Leu Ile His Gly Val Thr Arg Asn
5
<210>66
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>66
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaecatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtt cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240
ctgaagactg aggacgaggc tgactactac tgccagacgt acgaccccta cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>67
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>67
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Pro
85 90 95
Tyr Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>68
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>68
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>69
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>69
Ala Asn Asn Gln Arg Pro Ser
5
<210>70
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>70
Gln Thr Tyr Asp Pro Tyr Ser Val Val
5
<210>71
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>71
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
atttggggag tggctgggag ctggggccag gggacaatgg tcaccgtctc ctca 354
<210>72
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>72
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Trp Gly Val Ala Gly Ser Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210>73
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>73
Ser Tyr Ala Met Ser
5
<210>74
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>74
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>75
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>75
Asp Leu Ile Trp Gly Val Ala Gly Ser
5
<210>76
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>76
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtctt ataaccacaa ggacatcgtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>77
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>77
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asn His
85 90 95
Lys Asp Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>78
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>78
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>79
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>79
Ala Asn Asn Gln Arg Pro Ser
5
<210>80
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 08
<400>80
Gln Ser Tyr Asn His Lys Asp Ile Val
5
<210>81
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>81
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
attttcgggg tggggggggg gtggggccag gggacaatgg tcaccgtctc ctca 354
<210>82
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>82
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Phe Gly Val Gly Gly Gly Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210>83
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>83
Ser Tyr Ala Met Ser
5
<210>84
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>84
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>85
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>85
Asp Leu Ile Phe Gly Val Gly Gly Gly
5
<210>86
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>86
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcct actcccccca cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>87
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>87
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
His Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>88
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>88
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>89
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>89
Ala Asn Asn Gln Arg Pro Ser
5
<210>90
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 09
<400>90
Gln Ser Tyr Ser Pro His Ser Val Val
5
<210>91
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>91
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
attttcgggg tggggggggg gtggggccag gggacaatgg tcaccgtctc ctca 354
<210>92
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>92
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Phe Gly Val Gly Gly Gly Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210>93
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>93
Ser Tyr Ala Met Ser
5
<210>94
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>94
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>95
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>95
Asp Leu Ile Phe Gly Val Gly Gly Gly
5
<210>96
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>96
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcgt acgaccccca cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>97
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>97
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Pro
85 90 95
His Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>98
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>98
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>99
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>99
Ala Asn Asn Gln Arg Pro Ser
5
<210>100
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>100
Gln Ser Tyr Asp Pro His Ser Val Val
5
<210>101
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>101
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
atttggggag tggctgggag ctggggccag gggacaatgg tcaccgtctc ctca 354
<210>102
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>102
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Trp Gly Val Ala Gly Ser Trp Gly Gln Gly Thr
100 105 110
Met Val Thr ValSer Ser
115
<210>103
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>103
Ser Tyr Ala Met Ser
5
<210>104
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>104
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>105
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>105
Asp Leu Ile Trp Gly Val Ala Gly Ser
5
<210>106
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>106
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcct actcccccca cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>107
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>107
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
His Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>108
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>108
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>109
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>109
Ala Asn Asn Gln Arg Pro Ser
5
<210>110
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>110
Gln Ser Tyr Ser Pro His Ser Val Val
5
<210>111
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>111
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
attttcgggg tggggggggg gtggggccag gggacaatgg tcaccgtctc ctca 354
<210>112
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>112
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Phe Gly Val Gly Gly Gly Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210>113
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>113
Ser Tyr Ala Met Ser
5
<210>114
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>114
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>115
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>115
Asp Leu Ile Phe Gly Val Gly Gly Gly
5
<210>116
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>116
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagacgt acgaccccta cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>117
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>117
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Pro
85 90 95
Tyr Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>118
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>118
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>119
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>119
Ala Asn Asn Gln Arg Pro Ser
5
<210>120
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>120
Gln Thr Tyr Asp Pro Tyr Ser Val Val
5
<210>121
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>121
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
atttggggag tggctgggag ctggggccag gggacactgg tcaccgtctc ctca 354
<210>122
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>122
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Trp Gly Val Ala Gly Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>123
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>123
Ser Tyr Ala Met Ser
5
<210>124
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>124
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>125
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>125
Asp Leu Ile Trp Gly Val Ala Gly Ser
5
<210>126
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>126
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtctt atgacccgcg ggtggtcgtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>127
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>127
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Pro
85 90 95
Arg Val Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>128
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>128
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>129
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>129
Ala Asn Asn Gln Arg Pro Ser
5
<210>130
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>130
Gln Ser Tyr Asp Pro Arg Val Val Val
5
<210>131
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>131
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
atttggggag tggctgggag ctggggccag gggacactgg tcaccgtctc ctca 354
<210>132
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>132
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Trp Gly Val Ala Gly Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>133
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>133
Ser Tyr Ala Met Ser
5
<210>134
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>134
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>135
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>135
Asp Leu Ile Trp Gly Val Ala Gly Ser
5
<210>136
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>136
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtctt atgacccgac gaaccaggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>137
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>137
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Pro
85 90 95
Thr Asn Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>138
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>138
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>139
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>139
Ala Asn Asn Gln Arg Pro Ser
5
<210>140
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>140
Gln Ser Tyr Asp Pro Thr Asn Gln Val
5
<210>141
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>141
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
atttggggag tggctgggag ctggggccag gggacactgg tcaccgtctc ctca 354
<210>142
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>142
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Trp Gly Val Ala Gly Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210>143
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>143
Ser Tyr Ala Met Ser
5
<210>144
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>144
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>145
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>145
Asp Leu Ile Trp Gly Val Ala Gly Ser
5
<210>146
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>146
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcct actccccgac gagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>147
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>147
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
Thr Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>148
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>148
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>149
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>149
Ala Asn Asn Gln Arg Pro Ser
5
<210>150
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>150
Gln Ser Tyr Ser Pro Thr Ser Val Val
5
<210>151
<211>354
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>151
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aagagatcta 300
atttggggag tggctgggag ctggggccag gggacaatgg tcaccgtctc ctca 354
<210>152
<211>118
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>152
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Ile Trp Gly Val Ala Gly Ser Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210>153
<211>5
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>153
Ser Tyr Ala Met Ser
5
<210>154
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>154
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
5 10 15
Gly
<210>155
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>155
Asp Leu Ile Trp Gly Val Ala Gly Ser
5
<210>156
<211>336
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>156
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagacgt acgaccccta cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtgag 336
<210>157
<211>112
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>157
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Pro
85 90 95
Tyr Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glu
100 105 110
<210>158
<211>13
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>158
Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr Tyr Val Gln
5 10
<210>159
<211>7
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>159
Ala Asn Asn Gln Arg Pro Ser
5
<210>160
<211>9
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 16
<400>160
Gln Thr Tyr Asp Pro Tyr Ser Val Val
5
<210>161
<211>466
<212>DNA
＜213〉macaque (Macaca fascicularis)
<400>161
atgactcctg ggaagacctc attggtgcta ctgctgctgc tgctgagcct ggaggccata 60
gtgaaggcag gaatagcaat cccacgaaat tcaggatgcc caaattccga ggacaagaac 120
ttcccccgga ctgtgatggt caacctgaac atccataacc ggaataccag taccaatccc 180
aaaaggtcct cagattacta caaccgatcc acctcacctt ggaatctcca ccgcaatgag 240
gaccctgaga gatatccctc tgtgatctgg gaggcaaaat gccgccactt aggctgcgtc 300
aaggctgatg ggaacgtaga ctaccacatg aactctgtcc ccatccagca agagatcctg 360
gtcctgcgca gggagcctcg gcactgcccc aactccttcc ggctggagaa gatactggtg 420
tccgtgggct gcacctgtgt cacccccatt gtccaccatg tagcct 466
<210>162
<211>155
<212>PRT
＜213〉macaque (Macaca fascicularis)
<400>162
Met Thr Pro Gly Lys Thr Ser Leu Val Leu Leu Leu Leu Leu Leu Ser
1 5 10 15
Leu Glu Ala Ile Val Lys Ala Gly Ile Ala Ile Pro Arg Asn Ser Gly
20 25 30
Cys Pro Asn Ser Glu Asp Lys Asn Phe Pro Arg Thr Val Met Val Asn
35 40 45
Leu Asn Ile His Asn Arg Asn Thr Ser Thr Asn Pro Lys Arg Ser Ser
50 55 60
Asp Tyr Tyr Asn Arg Ser Thr Ser Pro Trp Asn Leu His Arg Asn Glu
65 70 75 80
Asp Pro Glu Arg Tyr Pro Ser Val Ile Trp Glu Ala Lys Cys Arg His
85 90 95
Leu Gly Cys Val Lys Ala Asp Gly Asn Val Asp Tyr His Met Asn Ser
100 105 110
Val Pro Ile Gln Gln Glu Ile Leu Val Leu Arg Arg Glu Pro Arg His
115 120 125
Cys Pro Asn Ser Phe Arg Leu Glu Lys Ile Leu Val Ser Val Gly Cys
130 135 140
Thr Cys Val Thr Pro Ile Val His His Val Ala
145 150 155
<210>163
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>163
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtctt atgatgacag cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>164
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 01
<400>164
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp
85 90 95
Ser Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>165
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>165
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtt cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240
ctgaagactg aggacgaggc tgactactac tgccagtcgt acgaccccca cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>166
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 02
<400>166
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Pro
85 90 95
His Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>167
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>167
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcct actcccccca cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>168
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 03
<400>168
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
His Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>169
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>169
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcct actccccgac gagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>170
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 04
<400>170
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
Thr Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>171
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>171
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtctt ataaccacaa ggacatcgtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>172
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 05
<400>172
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asn His
85 90 95
Lys Asp Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>173
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>173
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcgt acagcccgag cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>174
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 06
<400>174
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
Ser Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>175
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>175
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtt cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240
ctgaagactg aggacgaggc tgactactac tgccagacgt acgaccccta cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>176
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 07
<400>176
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Pro
85 90 95
Tyr Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>177
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>177
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcgt acgaccccca cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>178
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 10
<400>178
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Pro
85 90 95
His Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>179
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>179
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcct actcccccca cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>180
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 11
<400>180
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
His Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>181
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>181
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagacgt acgaccccta cagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>182
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 12
<400>182
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Pro
85 90 95
Tyr Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>183
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>183
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtctt atgacccgcg ggtggtcgtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>184
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 13
<400>184
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Pro
85 90 95
Arg Val Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>185
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>185
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtctt atgacccgac gaaccaggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>186
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 14
<400>186
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Pro
85 90 95
Thr Asn Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>187
<211>330
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>187
aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60
tcctgcaccc gcagcagtgg cagccttgcc aactactatg tgcagtggta ccaacagcgc 120
ccgggcagtg cccccaccat tgtgatcttt gcgaataacc aaagaccctc tggggtccct 180
gatcgattct ctggctccat cgacagctcc tccaactctg cctccctcac catctctaga 240
ctgaagactg aggacgaggc tgactactac tgccagtcct actccccgac gagcgtggtg 300
ttcggcggag ggaccaagct gaccgtccta 330
<210>188
<211>110
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉antibody 15
<400>188
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Leu Ala Asn Tyr
20 25 30
Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val
35 40 45
Ile Phe Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Arg
65 70 75 80
Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Pro
85 90 95
Thr Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210>189
<211>30
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉the VH framework region 1
<400>189
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30
<210>190
<211>14
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉the VH framework region 2
<400>190
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
1 5 10
<210>191
<211>32
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉the VH framework region 3
<400>191
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210>192
<211>11
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉the VH framework region 4
<400>192
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210>193
<211>22
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉the VL framework region 1
<400>193
Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
1 5 10 15
Thr Val Thr Ile Ser Cys
20
<210>194
<211>15
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉the VL framework region 2
<400>194
Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Ile Val Ile Phe
1 5 10 15
<210>195
<211>34
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉the VL framework region 3
<400>195
Gly Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Ser Ser Ser Asn Ser
1 5 10 15
Ala Ser Leu Thr Ile Ser Gly Leu Lys Thr Glu Asp Glu Ala Asp Tyr
20 25 30
Tyr Cys
<210>196
<211>10
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉the VL framework region 4
<400>196
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
1 5 10
<210>197
<211>153
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic sequence: the end-labelled human il-17 A of N-
<400>197
Met Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His
1 5 10 15
Glu Ile Val Lys Ala Gly Ile Thr Ile Pro Arg Asn Pro Gly Cys Pro
20 25 30
Asn Ser Glu Asp Lys Asn Phe Pro Arg Thr Val Met Val Asn Leu Asn
35 40 45
Ile His Asn Arg Asn Thr Asn Thr Asn Pro Lys Arg Ser Ser Asp Tyr
50 55 60
Tyr Asn Arg Ser Thr Ser Pro Trp Asn Leu His Arg Asn Glu Asp Pro
65 70 75 80
Glu Arg Tyr Pro Ser Val Ile Trp Glu Ala Lys Cys Arg His Leu Gly
85 90 95
Cys Ile Asn Ala Asp Gly Asn Val Asp Tyr His Met Asn Ser Val Pro
100 105 110
Ile Gln Gln Glu Ile Leu Val Leu Arg Arg Glu Pro Pro His Cys Pro
115 120 125
Asn Ser Phe Arg Leu Glu Lys Ile Leu Val Ser Val Gly Cys Thr Cys
130 135 140
Val Thr Pro Ile Val His His Val Ala
145 150
<210>198
<211>132
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉ripe human il-17 A
<400>198
Gly Ile Thr Ile Pro Arg Asn Pro Gly Cys Pro Asn Ser Glu Asp Lys
1 5 10 15
Asn Phe Pro Arg Thr Val Met Val Asn Leu Asn Ile His Asn Arg Asn
20 25 30
Thr Asn Thr Asn Pro Lys Arg Ser Ser Asp Tyr Tyr Asn Arg Ser Thr
35 40 45
Ser Pro Trp Asn Leu His Arg Asn Glu Asp Pro Glu Arg Tyr Pro Ser
50 55 60
Val Ile Trp Glu Ala Lys Cys Arg His Leu Gly Cys Ile Asn Ala Asp
65 70 75 80
Gly Asn Val Asp Tyr His Met Asn Ser Val Pro Ile Gln Gln Glu Ile
85 90 95
Leu Val Leu Arg Arg Glu Pro Pro His Cys Pro Asn Ser Phe Arg Leu
100 105 110
Glu Lys Ile Leu Val Ser Val Gly Cys Thr Cys Val Thr Pro Ile Val
115 120 125
His His Val Ala
130
<210>199
<211>17
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>
＜223〉human il-17 A peptide
<400>199
Cys Arg His Leu Gly Cys Ile Asn Ala Asp Gly Asn Val Asp Tyr His
1 5 10 15
Met
<210>200
<211>160
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic sequence: the mutant IL-17A precursor that contains the His label
<400>200
Met Thr Pro Gly Lys Thr Ser Leu Val Ser Leu Leu Leu Leu Leu Ser
1 5 10 15
Leu Glu Ala Ile Val Lys Ala Gly Ile Thr Ile Pro Arg Asn Pro Gly
20 25 30
Cys Pro Asn Ser Glu Asp Lys Asn Phe Pro Arg Thr Val Met Val Asn
35 40 45
Leu Asn Ile His Asn Arg Asn Thr Asn Thr Asn Pro Lys Arg Ser Ser
50 55 60
Asp Tyr Tyr Asn Arg Ser Thr Ser Pro Trp Asn Leu His Arg Asn Glu
65 70 75 80
Asp Pro Glu Arg Tyr Pro Ser Val Ile Trp Glu Ala Lys Cys Arg His
85 90 95
Gln Arg Cys Val Asn Ala Glu Gly Lys Leu Asp His His Met Asn Ser
100 105 110
Val Pro Ile Gln Gln Glu Ile Leu Val Leu Arg Arg Glu Pro Pro His
115 120 125
Cys Pro Asn Ser Phe Arg Leu Glu Lys Ile Leu Val Ser Val Gly Cys
130 135 140
Thr Cys Val Thr Pro Ile Val His His Val Ala His His His His His
145 150 155 160
<210>201
<211>132
<212>PRT
＜213〉artificial sequence
<220>
＜223〉synthetic sequence: the IL-17A of mutant maturation
<400>201
Gly Ile Thr Ile Pro Arg Asn Pro Gly Cys Pro Asn Ser Glu Asp Lys
1 5 10 15
Asn Phe Pro Arg Thr Val Met Val Asn Leu Asn Ile His Asn Arg Asn
20 25 30
Thr Asn Thr Asn Pro Lys Arg Ser Ser Asp Tyr Tyr Asn Arg Ser Thr
35 40 45
Ser Pro Trp Asn Leu His Arg Asn Glu Asp Pro Glu Arg Tyr Pro Ser
50 55 60
Val Ile Trp Glu Ala Lys Cys Arg His Gln Arg Cys Val Asn Ala Glu
65 70 75 80
Gly Lys Leu Asp His His Met Asn Ser Val Pro Ile Gln Gln Glu Ile
85 90 95
Leu Val Leu Arg Arg Glu Pro Pro His Cys Pro Asn Ser Phe Arg Leu
100 105 110
Glu Lys Ile Leu Val Ser Val Gly Cys Thr Cys Val Thr Pro Ile Val
115 120 125
His His Val Ala
130
Claims (15)
1. the antibody of the human il-17 A of a separation, wherein, the variable region of heavy chain of described antibody and variable region of light chain comprise hexad CDR, wherein the CDR1 to CDR3 of heavy chain is SEQ ID NO:63, SEQ IDNO:64 and SEQ ID NO:65, and the CDR1 to CDR3 of light chain is SEQ ID NO:68, SEQ IDNO:69 and SEQ ID NO:70.
2. antibody as claimed in claim 1 is characterized in that, the IC50 that discharges the described antibody of measuring in the HT1080 test cell line of IL-6 in that 1nM human il-17 A is reacted is no more than 1nM.
3. antibody as claimed in claim 1 is characterized in that, the IC50 of the described antibody of measuring in the HT1080 test cell line of the collaborative IL-6 of release in that 125pM human il-17 A and 25pM TNF α are reacted is no more than 0.5nM.
4. the antibody of a separation, it comprises VH structural domain and aminoacid sequence the VL structural domain as SEQ ID NO:176 shown in of aminoacid sequence shown in SEQ ID NO:62.
5. antibody as claimed in claim 4 is characterized in that, described antibody molecule is IgG1.
6. antibody as claimed in claim 4 is characterized in that, described antibody molecule is scFv.
7. composition, it comprises such as each described antibody among the claim 1-6, and pharmaceutically acceptable vehicle.
8. be used at the composition by surgical operation or therapy processes human or animal body, comprise such as each described antibody among the claim 1-6.
9. the application of each described composition in preparing the medicine for the treatment of the disease that is selected from rheumatoid arthritis and osteoarthritis in each described antibody or claim 7 and 8 among the claim 1-6.
10. application as claimed in claim 9 is characterized in that, described disease is rheumatoid arthritis.
11. the nucleic acid molecule of a separation, each described antibody among its coding claim 1-6.
12. one kind with the host cell of nucleic acid vitro conversion as claimed in claim 11.
13. a method that produces antibody, the method are included in and cultivate host cell as claimed in claim 12 under the condition that produces described antibody.
14. method as claimed in claim 13 is characterized in that, also comprises separating and/or the described antibody of purifying.
15. a generation comprises the method for the composition of antibody, the method comprises:
Produce antibody by method as claimed in claim 14; With
Described antibody is mixed with the composition that comprises at least a other component.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US81582806P | 2006-06-23 | 2006-06-23 | |
| US60/815,828 | 2006-06-23 | ||
| US91356607P | 2007-04-24 | 2007-04-24 | |
| US60/913,566 | 2007-04-24 | ||
| PCT/SE2007/000600 WO2007149032A1 (en) | 2006-06-23 | 2007-06-20 | Antibody molecules for human il-17 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101611057A CN101611057A (en) | 2009-12-23 |
| CN101611057B true CN101611057B (en) | 2013-03-20 |
Family
ID=38833679
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007800235664A Expired - Fee Related CN101611057B (en) | 2006-06-23 | 2007-06-20 | Antibody molecules for human il-17 |
Country Status (13)
| Country | Link |
|---|---|
| US (1) | US8865166B2 (en) |
| EP (1) | EP2041176B1 (en) |
| JP (1) | JP5385784B2 (en) |
| KR (1) | KR20090029272A (en) |
| CN (1) | CN101611057B (en) |
| AR (1) | AR061568A1 (en) |
| AU (1) | AU2007261748B2 (en) |
| BR (1) | BRPI0713382A2 (en) |
| CA (1) | CA2655976A1 (en) |
| ES (1) | ES2399475T3 (en) |
| TW (1) | TW200815469A (en) |
| UY (1) | UY30430A1 (en) |
| WO (1) | WO2007149032A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112203685A (en) * | 2018-03-29 | 2021-01-08 | 瑞美德生物医药科技有限公司 | Treatment of autoimmune and inflammatory disorders using antibodies that bind interleukin-17A (IL-17A) |
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| DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
| AU2006284841B2 (en) * | 2005-09-01 | 2012-11-08 | Merck Sharp & Dohme Corp. | Use of IL-23 and IL-17 antagonists to treat autoimmune ocular inflammatory disease |
| JP2009513708A (en) | 2005-10-31 | 2009-04-02 | オンコメッド ファーマシューティカルズ インコーポレイテッド | Compositions and methods for diagnosis and treatment of cancer |
| US7723477B2 (en) | 2005-10-31 | 2010-05-25 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for inhibiting Wnt-dependent solid tumor cell growth |
| AU2006319358B2 (en) | 2005-11-30 | 2012-01-19 | AbbVie Deutschland GmbH & Co. KG | Anti-Abeta globulomer antibodies, antigen-binding moieties thereof, corresponding hybridomas, nucleic acids, vectors, host cells, methods of producing said antibodies, compositions comprising said antibodies, uses of said antibodies and methods of using said antibodies |
| KR101439828B1 (en) | 2005-11-30 | 2014-09-17 | 애브비 인코포레이티드 | Monoclonal antibodies against amyloid beta protein and uses thereof |
| US7910703B2 (en) | 2006-03-10 | 2011-03-22 | Zymogenetics, Inc. | Antagonists to IL-17A, IL-17F, and IL-23P19 and methods of use |
| AU2007226627B2 (en) | 2006-03-10 | 2012-09-20 | Zymogenetics, Inc. | Antibodies that bind both IL-17A and IL-17F and methods of using the same |
| US7790163B2 (en) | 2006-03-10 | 2010-09-07 | Zymogenetics, Inc. | Antibodies that bind both IL-17A and IL-17F and methods of using the same |
| GB0612928D0 (en) | 2006-06-29 | 2006-08-09 | Ucb Sa | Biological products |
| WO2008021156A2 (en) | 2006-08-11 | 2008-02-21 | Schering Corporation | Antibodies to il-17a |
| GB0620729D0 (en) | 2006-10-18 | 2006-11-29 | Ucb Sa | Biological products |
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| BRPI0713382A2 (en) | 2012-05-29 |
| TW200815469A (en) | 2008-04-01 |
| CN101611057A (en) | 2009-12-23 |
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| CA2655976A1 (en) | 2007-12-27 |
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| AU2007261748A1 (en) | 2007-12-27 |
| WO2007149032A1 (en) | 2007-12-27 |
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